abstract: We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization/recellularization and a combination of stem cells and tissue engineering.
author: Yunying Liu; Ru Yang; Zuping He; Wei-Qiang Gao
date: 2013-01-22
references:
title: Generation of functional organs from stem cells

# Introduction

Stem cells are undifferentiated cells found in the body which have the ability to continuously divide, self-renew themselves and differentiate into various kinds of cells. With the capability of self-renewal, pluripotency and differentiation, stem cells have been believed to be useful for treatment of a wide variety of diseases in the future, including stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, spinal cord injury, baldness, blindness, deafness, wound healing, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, osteoarthritis rheumatoid arthritis, Crohn's disease, and diabetes. Amongst the applications, a number of adult stem cell therapies have already been practiced clinically. As an example, hematopoietic stem cell transplantation has been successfully applied to treat leukemia.

In addition to cell replacement therapy using stem cells, organ transplantation has been successfully practiced in clinics for organ failure of the liver or kidney. However, the severe shortage of donor organs has become the main obstacle to expand the organ transplant program. Generation of biological or semi-biological organs could be an alternative approach to solve the problem of the donor organ shortage. Notably, researchers have been hunting for ways to establish a whole organ using stem cells.

In recent years encouraging approaches for functional organ generation have emerged. The present manuscript provides an overview of organ generation using a single adult tissue stem cell, a blastocyst complementation system coupled with a specific stem cell niche, a method of decellularization and recellularization of bio-scaffold, and a combinatorial approach of tissue engineering and stem cells.

## Generation of a functional organ from a single adult tissue stem cell

To demonstrate whether there are true stem cells in a given tissue, one needs to show that a single stem cell purified from the tissue has the capability of generating the entire organ. Up to present, it has been elegantly shown by independent groups that the mammary gland and the prostate can be generated in vivo from a single adult tissue stem cell \[1–3\].

Two laboratories have reported independently that single stem cells isolated from adult mouse mammary glands are able to produce secretory mammary glands when they are transplanted in the fat-pad in mice \[1, 2\]. It is long believed that there are stem cells in the mammary glands because this organ has the capability of undergoing an extensive growth at puberty and a second phase of expansion and retraction during pregnancy under the regulation of estrogen \[4\]. However, due to the lack of defined markers, there has been no reliable method to isolate mammary stem cells. This hypothesis was not proven until the work by the two groups. Based on some previous work by Clark and colleague \[5\], Shackleton et al. isolated putative mouse mammary stem cells using specific cell-surface markers (Lin^-^, CD29^hi^, CD24^+^) by FACS. They demonstrated these Lin^-^CD29^hi^CD24^+^ mammary cells have *in vitro* sphere formation capability and the ability to repopulate all mammary epithelial cells after transplantation into the fat-pad. Importantly, the investigators employed a lineage-tracer in the stem cells so to follow their ultimate phenotypes *in vivo*. They showed elegantly that a single cell within the Lin^-^CD29^hi^CD24^+^ population of the mouse mammary gland, marked with a LacZ report transgene, can reconstitute a completely functional mammary gland *in vivo*. Notably, the transplanted cell contributes to both the luminal and myoepithelial lineages and generates milk-producing lobuloalveolar units during pregnancy. This is the first report to demonstrate that a single adult tissue stem cell has multi-lineage differentiation capacity to produce a functional organ in an *in vivo* setting.

Similarly, using a colony-formation *in vivo* assay and an *in vivo* renal capsule transplantation approach, Gao and his colleagues have also reported that a single stem cell isolated from the adult mouse prostate epithelium has the capacity to generate a functional prostate \[3\]. Actually, prostate stem cells were postulated to exist because of its regenerative feature following androgen deprivation and replacement more than twenty years ago \[6\]. Because cell death mainly occurs in the luminal cell compartment after castration and cell proliferation mainly happens in the basal compartment following androgen replacement, stem cells are generally believed to reside in basal cell compartment and are able to repopulate the entire prostate epithelial cells.

First, this group identified a new marker for prostate stem cells, CD117, based on the following 6 features: 1) It is enriched in proximal region of the prostate; 2) It is mainly expressed in basal cell population; 3) It is upregulated after castration and returned to normal levels following androgen replacement; 4) Only CD117^+^ cells, but not the CD117^-^ cells, can form colony structures with lumen *in vitro*; 5) It can generate prostate epithelial glandular structures *in vivo*; 6) CD117+ cells from 2^nd^ and 3^rd^ generation grafts also have the self-renewal capability.

To demonstrate whether a single stem cell has the ability to generate a prostate structure, the investigators first further enriched the stem cell population by sorting the cells expressing multiple stem cell markers, Sca-1^+^CD133^+^CD44^+^CD117^+^, into individual wells of a 96-well plate, verified them under a microscope, and transplanted them in a combination with rat embryonic urogenital sinus mesenchymal cells (rUGM) under the renal capsule of athymic nu/nu mouse hosts. Three months later, they removed the kidneys and analyzed the fate of the grafted cells. Of the 97 single-cell transplants, there were about 1/7 Sca-1^+^CD133^+^CD44^+^CD117^+^ grafts that demonstrate a branching pattern with epithelial tubules composed of prostatic basal, luminal and neuroendocrine lineages by the histological and immunocytochemical examination.

The generation of mammary gland and prostate acini from single stem cell implants in mice is a major breakthrough. These findings raise the possibility that people who have lost their mammary glands or prostates due to cancer could grow new ones. On the other hand, it is believed that in some tissues, there are cancer stem cells that are tumor-initiating cells. Cancer stem cells might be derived from normal stem cells in which specific tumor suppressor genes are mutated or lost \[7\]. Therefore, although the prostate glands were grown in mice but the research may aid the identification of markers for cancer stem cells, which may help diagnosis and more efficient treatment of prostate cancer in humans. These studies on reconstruction of mammary and prostate glands using single adult stem cells have important ramifications not only for tissue repair/regeneration, but also for identification of mammary and prostate cancer stem cells. In other words, we envision that the potential use of single stem cells in clinic will ultimately change the treatment paradigm for human disorders more than mammary gland injury and prostatic disease.

## Generation of organs using a blastocyst complementation system

In addition to single stem cells prepared from specific adult tissue, embryonic stem cells (ESCs) have been shown to be able to produce specific organs by using a strategy of injection of ESCs from one species into the blastocyst of another species. The development of a specific organ can be precluded by genetic manipulation in the recipient species but still providing a niche for organ development. The pluripotent stem cell-derived cells from the donor species would then developmentally compensate for the defect and produce the missing organ.

The blastocyst complementation system was first reported by Chen et al. by implanting normal mouse embryonic stem cells (mESCs) into the blastocysts derived from Rag2^−/−^ mice to generate T and B lymphocyte lineages \[8\]. In recent years, this system was applied to generate pluripotent stem cell (PSC)–derived donor organs *in vivo* due to the fact that complex cellular interactions among and within tissues that are required for organogenesis are difficult to recapitulate *in vitro*. For this purpose, PSCs were injected into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation just to supply as a niche for organ development. The PSC-derived cells would form the missing organ as a result of developmental compensation for the defect.

Kobayashi and colleagues had showed proof-of-principle findings of pancreas generation by injection of PSCs into the blastocysts of pancreas-deficient *Pdx1* ^−/−^ mouse (pancreatogenesis-disabled) \[9\]. More specifically, they injected induced pluripotent stem cells (iPS) taken from rats into the blastocysts of mice that were unable to grow their own pancreas and were unable to produce insulin. The rat stem cells grew in the special mouse environment in the absent of mouse pancreas. When the mice matured to adulthood, they developed a rat pancreas from the injected rat stem cells. The origin and function of pancreatic cells were analyzed in the PSC-derived rat pancreas in adult mice. Glucose load was tested and the mice showed no signs of diabetes. These findings clearly indicate that functional rat pancreas are successfully generated in *Pdx1* ^−/−^ mice using the inter-species blastocyst complementation.

The same technique has been also used to generate kidney using *Sall1* ^−/−^ mouse blastocysts \[10\]. Rat ESC or PSC are injected into *Sall1* ^−/−^ mouse blastocysts and the entire rat kidneys are formed by the injected mouse ESC- or PSC-derived cells. These ESC/PSC-derived kidneys connect with ureters and function well. The bladders are full of urine.

Generation of pancreas and kidney via blastocyst complementation is an innovative approach. It demonstrates that this technique can be used to generate complex organ using donor PSCs. With an empty developmental niche, a solid organ will be produced developmentally from PSCs-derived cellular progeny in the vacant space.

The current organ shortage means that patients must endure a long time on waiting lists for transplantation. This blastocyst complementation system may therefore provide a novel approach for organ supply. The ultimate goal of blastocyst complementation system is to generate specific human organs from various pluripotent stem cells. However, such inter-species technology using human stem cells involves ethical issues which has to meet the scientific and ethical guidelines proposed by the International Society for Stem Cell Research (ISSCR) and requires approval from related ethical committees.

## Engineering a whole organ via decellularization of matrix bioscaffolds and recellularization with stem cells

Organ formation requires not only stem cells but also the surrounding stem cell niche or microenvironment and extracellular matrix participation. In fact, three-dimensional tissue scaffold has been demonstrated to be very critical for organ regeneration.

Decellularization and recellularization of matrix bioscaffolds have been shown to be a promising approach for whole-organ tissue engineering in recent years \[11\]. Donor organs such as the heart \[12\], liver \[13\], lung \[14\], kidney \[15\] and bladder \[16\] can be decellularized to an acellular biologic scaffold material and then be recellularized with functional parenchymal cells and/or selected progenitor cell populations. Encouraging results have emerged in animal model studies.

Taylor and colleagues have generated a bioartificial heart which has a heart structure with appropriate cell composition and cardiac pump function using this attractive technique in 2008 \[12\]. They perfused heart with 1% SDS, a strong ionic detergent, to produce acellular scaffolds, preserved extracellular matrix and original microvascular network, and then repopulated these heart-like constructs with neonatal cardiomyocytes and aortic endothelial cells. When proper physiological stimuli were given, the recellularized organ in the culture was able to further mature and display beating behaviors. Rhythmic contractions were observed by day 4, pump function generated by day 8 and cardiac architecture was formed, as confirmed by histological analysis. The bioartificial heart pumped with an amazing function, which was equivalent to about 2% heart power of an adult or 25% heart function of a 16-week fetus.

Uygun and colleagues modified Taylor's perfusion decellularization technique to achieve transplantable liver grafts in 2010 \[13\]. The ischemic livers were perfused through the portal vein with SDS. The intact lobular structure and vascular network were retained in the decellularized liver bioscaffold. The reseeded adult hepatocytes and nonparenchymal cells were able to repopulate to create a recellularized whole liver graft. The graft was transplanted as auxiliary heterotopic graft with portal vein arterializations and animal was shown to be viable for 8 hours after transplantation. The hepatocytes in the recellularized liver graft were metabolically active with functions of urea and albumin secretion. Similarly, Niklason and colleagues regenerated lung grafts using the decellularization and recellularization strategy in 2010 \[14\]. They removed cellular components from lung matrix, preserved hierarchical branching structures of the airways and vasculature bed in the extracellular scaffold, and reseeded pulmonary and vascular endothelial cells in the acellular lung matrix. The engineered lungs presented pneumonocyte viability and function after implantation, as evidenced by oxygen exchange.

These exciting studies on complex organs, such as the heart, liver and lung as described above, provide insights into the power of the methodology. The technology has multiple advantages. First, a bioartificial organ could theoretically solve the problem of lifelong immunosuppression after organ transplantation. Second, the preserved extracellular matrix and three-dimensional scaffold provide important signals for engraftment of repopulated cells, survival of engrafted cells, and function of newborn organ. Third, the vascular bed in the decellularized bioscaffold allows oxygen and nutrient rapid to be delivered after recellularization and reconnection to the circulation.

The most important concern in the technology is the cell type and the source of cells used for filling the decellularized organ matrix. Different types of cells are needed to reconstruct the parenchyma, vasculature, and support structures underneath. An ideal strategy is that a stem or progenitor cell can self-renew and differentiate into heterogeneous types of cells as needed to form a functional organ. Currently, cell sources are broadly categorized. Stem or progenitor cells used for recellularization include embryonic stem cells (ESCs), fetal cells, adult-derived stem or progenitor cells and adult tissue-derived inducible pluripotent stem cells (iPSCs). Non-stem or progenitor cells used for repopulation are usually parenchymal cells (e.g. cardiomyocytes, hepatocytes, pneumonocytes), vascular cells (e.g. endothelial cells) and supportive cells (e.g. fibroblasts). Whether autologous or allogeneic cells are utilized will depend on cell numbers required and if the need is immediate.

In short, decellularization and recellularization technique is a potential breakthrough in whole-organ tissue engineering. With further development, organs (e.g. heart, liver, lung, kidney, pancreas) can be stripped of their cells and regrow to pump blood, to generate albumin and urea, to exchange oxygen to produce urine and to secret insulin/glucagon with replaced cardiocytes, hepatocytes, alveolar type 2 cells, renal medulla cells and islet α, β cells.

## Generation of organs by a combination of tissue-engineering and seeded stem cells

Tissue-engineering techniques have been used to combine specific stem cells with biocompatible and biodegradable polymer scaffolds to produce a cell polymer implant. The field of tissue engineering has been developed to solve the problem of donor-tissue scarcity either as whole organs or as reconstructive grafts, such as cartilage frameworks for total ear reconstruction \[17, 18\]. Cao and colleagues were the first to apply polyglycolic acid-polylactic acid to form a polymer template in the shape of a human auricle and seeded chondrocytes into the construct. New tissue-engineered cartilage grew for 12 weeks after implantation under the dorsal skin of mice. The polyglycolic acid-polylactic acid construct degraded gradually following transplantation, but the cartilage was formed and confirmed by morphological and histological analysis \[17\]. The technique is particularly useful for plastic and reconstructive surgery.

But this system, for quite a long time, seems mainly focused on the exterior instead of the interior of the function of the organ because of the key challenge to integrate fully functioning vascular architectures into the engineered construct. Therefore, the generation of functional human vascular network by Takebe and colleagues provides an innovative strategy to regenerate larger, well-vascularized organs including the liver and seems to solve the major obstacles in regenerating thick, complex tissues. They co-cultured human umbilical vein endothelial cells and mesenchymal stem cells in a matrix made of collagen and fibronectin. The human umbilical vein endothelial cells were labeled with GFP and the human mesenchymal stem cells were with kusabira orange. Vessel-like constructs were formed and detected under a fluorescence microscope *in vitro* seven days after cultivation. More importantly, when the vascular network constructs were implanted into an immunodeficient mouse, they were able to infuse the structures with rhodamine dextran to confirm that the structures can act as functional vessels. Study in this direction will facilitate future reconstitution of vascularized human organs \[19\].

# Conclusions

In summary, as described above, there are four major ways of generation of functional organs using stem cells (Figure 1). Each of them has its own uniqueness. Specifically, using adult stem cells such as adult tissue epithelial stem cells, there will be a very low risk of tumorigenesis, more selective differentiation capability and better integration into the related tissue mass and acquisition of relevant organ functions. However, the number of the adult tissue stem cells that can be isolated is a limiting factor. Although using blastocyst complementation system is an attractive approach, particularly using a human-porcine inter-species strategy, injection of human stem cells into an organ-deficient porcine embryo is currently forbidden for ethical reasons. In fact, blastocyst complementation across species of large livestock animals such as porcine and canine has not been experimentally proven, more studies are needed in this direction. While the decellularization and recellularization research on various types of organs including the heart, liver, lung, kidney, bladder, etc. has yielded exciting results, but it is not practical to get the donor organs for such approach from the human beings. In addition, the acquisition of the organ functions from these studies is still limited. Tissue engineering using various biomaterial scaffolds holds hopes for particular medical needs such as plastic and reconstructive surgery, however the scaffolds using particular biomaterials are still primitive and require further development and research. Finally, the stem cell types that can be considered to be used for generation of functional organs may include ESC, iPSC and adult tissue cells. Each of these cell types has different advantages and disadvantages, in terms of proliferation, differentiation and tumorigenicity risk, which have been well-discussed in the literature.

Although organogenesis is a complex process, the organ generation systems described above using stem cells or a combination of stem cells and tissue engineering may be applied, or at least raise the hope, to treat organ failure in humans in the near future. Besides adult tissue stem cells and embryonic stem cells, recent burgeoning and promising development of technology of induced pluripotent stem cells opens a new avenue for potential cell replacement and organ generation. Relevant to generation of functional organs, it is worth-mentioning that functional hepatocyte-like cells can be generated from induced pluripotent stem cells and the liver can be partially reconstituted in Fah−/− mice \[20\].

Taken together, with the above initial steps and further improvement, stem cell therapy may one day not only repair tissue damage but also generate new tissues for tissue/organ transplantation. Even though it is still at an infancy stage, these studies may hold promise for generation of specific functional organs for organ transplantation, to help solve the clinical problem of donor shortage.

## Acknowledgements

The study is supported by funds from the Chinese Ministry of Science and Technology (2012CB966800 and 2013CB945600 to WQG and RY and 2012CB967900), the National Natural Science Foundation of China (81130038 to WQG, 31171422 and 31230048 to ZH), Science and Technology Commission of Shanghai Municipality (Pujiang program to WQG, 11PJ1406400 to ZH), Shanghai Education Committee Key Disciplines and Specialties Foundation (J50208 to WQG), Shanghai Health Bureau Key Disciplines and Specialties Foundation (to WQG), KC Wong foundation (to WQG) and the China Postdoctoral Science Foundation (2012M510835 to YYL).

# References


abstract: # OBJECTIVE
  .
  Time-related biases in observational studies of drug effects have been described extensively in different therapeutic areas but less so in diabetes. Immortal time bias, time-window bias, and time-lag bias all tend to greatly exaggerate the benefits observed with a drug.
  .
  # RESEARCH DESIGN AND METHODS
  .
  These time-related biases are described and shown to be prominent in observational studies that have associated metformin with impressive reductions in the incidence of and mortality from cancer. As a consequence, metformin received much attention as a potential anticancer agent; these observational studies sparked the conduction of randomized, controlled trials of metformin as cancer treatment. However, the spectacular effects reported in these studies are compatible with time-related biases.
  .
  # RESULTS
  .
  We found that 13 observational studies suffered from immortal time bias; 9 studies had not considered time-window bias, whereas other studies did not consider inherent time-lagging issues when comparing the first-line treatment metformin with second- or third-line treatments. These studies, subject to time-related biases that are avoidable with proper study design and data analysis, led to illusory extraordinarily significant effects, with reductions in cancer risk with metformin ranging from 20 to 94%. Three studies that avoided these biases reported no effect of metformin use on cancer incidence.
  .
  # CONCLUSIONS
  .
  Although observational studies are important to better understand the effects of drugs, their proper design and analysis is essential to avoid major time-related biases. With respect to metformin, the scientific evidence of its potential beneficial effects on cancer would need to be reassessed critically before embarking on further long and expensive trials.
author: Samy Suissa; Laurent AzoulayCorresponding author: Samy Suissa, <email@example.com>.
date: 2012-12
references:
subtitle: : Time-related biases in observational studies
title: Metformin and the Risk of Cancer

Time-related biases are common in observational studies of drug effects and have been described extensively in several therapeutic areas. However, these biases have not been described in detail in the field of diabetes. Time-related biases include immortal time bias, a bias introduced with time-fixed cohort analyses that misclassify unexposed time as exposed; time-window bias, a bias introduced because of differential exposure opportunity time windows between subjects; and time-lag bias, a bias introduced by comparing treatments given at different stages of the disease (1,2). These biases are known to exaggerate downward the effect of a drug, thus making a drug seem to be protective when in fact it may have no effect. In this review, we show that several of the observational studies investigating the association between metformin and cancer incidence and mortality are affected by these time-related biases.

Metformin is a drug of choice for the management of type 2 diabetes mellitus (3,4). It reduces insulin resistance, improves glycemic control, and can be combined safely with other antidiabetic drugs (5). In 2005, an observational study using data from Tayside, Scotland, reported a significant 23% reduction in the incidence of any cancer with metformin use, thus putting forward the hypothesis that metformin could lower the risk of cancer onset in patients with diabetes (6). This study generated great interest in metformin as an agent in the prevention and treatment of cancer, and many preclinical studies showed that metformin can inhibit the growth of cancer cells in vitro and in vivo (7,8).

In parallel, a series of observational studies conducted in various databases generally reported similar beneficial results with metformin, thus "confirming" the findings of the 2005 study. Two meta-analyses including some of these observational studies reported a highly significant reduction in cancer incidence or mortality associated with metformin use (risk reductions ranged between 31 and 34%) (9,10). This convergence of evidence from multiple preclinical and epidemiological studies formed the impetus to recommend the conduct of randomized, controlled trials (RCTs) of metformin in the prevention and treatment of cancer (11–14). However, as discussed later, several of these observational studies had important time-related biases that likely exaggerated the potential antitumor effects of metformin.

# STUDIES OF CANCER PREVENTION

The majority of studies published to date have reported the role of metformin in preventing the occurrence of cancer in general and of some cancers in particular. To describe the different variations of this bias, we use only some of these studies as a model; the list of studies that incurred the same biases is provided in Table 1 (15–37).

Time-related biases in observational studies investigating the effects of metformin on cancer incidence and as a cancer treatment

![](2665tbl1)

## Immortal time bias

The first published epidemiological study to verify this hypothesis involved a cohort of patients of 10,309 new users of oral hypoglycemic agents (OHAs) identified from the Saskatchewan Health databases (15). Patients entered the cohort at the time of their first OHA prescription during 1991–1996. They were followed through 1999 for death due to cancer, which occurred in 407 patients. Exposure was classified as sulfonylureas only or metformin, with the latter including users of only metformin and those who also used sulfonylureas at some point in time during follow-up. A Cox proportional hazards model was used to estimate the hazard ratio (HR), adjusted for age, sex, insulin use, and a chronic disease score. The adjusted HR of cancer mortality for sulfonylurea versus metformin was 1.3 (95% CI 1.1–1.6), which, when reversed, gives the HR for metformin relative to sulfonylurea as 0.8 (0.6–0.9), suggesting a protective effect of metformin.

Immortal time bias is introduced in this study from its definition of exposure and related analysis. The 3,340 subjects who entered the cohort while using sulfonylureas and remained on these throughout the entire follow-up and the 1,229 who used only metformin from day 1 throughout follow-up pose no problem to the definition of exposure. The bias is introduced by classifying some of the remaining 5,740 who used sulfonylureas and metformin at various times during follow-up as "metformin users." Although those whose cohort entry prescription was metformin are classified correctly in the metformin-exposed group, those whose cohort entry prescription was a sulfonylurea are not (Fig. 1). Indeed, the time between the sulfonylurea prescription at entry into the cohort and the first metformin prescription during follow-up is called "immortal" (thick gray line in Fig. 1) because the patient must be alive to have received their first metformin prescription (1). Moreover, this immortal person-time should be classified as sulfonylurea-exposed until the start of metformin, at which point the remaining person-time can be classified as metformin-exposed. In this study, the authors misclassified this immortal person-time as metformin-exposed, which will lead to immortal time bias.

Because the data necessary to assess the magnitude of the bias are not provided in the article, for the sake of illustration we assume that the mean delay between cohort entry and the first metformin prescription was 1 year among the 5,740 combined users. This would result in 5,740 immortal person-years of sulfonylurea exposure misclassified as metformin exposure. The reported rates of cancer death for metformin (245/39,026 = 6.3 per 1,000 person-years) and for sulfonylureas (162/16,700 = 9.7 per 1,000 person-years), which were based on misclassified denominators, produced a crude rate ratio of 0.65. By properly reclassifying exposure (per our assumption), the rates would become 245/(39,026–5,740) = 7.4 per 1,000 person-years for metformin and 162/(16,700+5,740) = 7.2 per 1,000 person-years for sulfonylureas, resulting in a corrected crude rate ratio of 1.0. There are more sophisticated ways to estimate the rate ratio while properly accounting for such time-varying exposures, such as Cox proportional hazards models with time-dependent factors. An alternative approach simply is to use an intent-to-treat approach (as is done in RCTs) that classifies exposure according to the first OHA prescription type, irrespective of switching to or adding the other agent, with, of course, the limitations of interpretation inherent to this approach.

In a subsequent publication reanalyzing the same data, the authors recognized the presence of immortal time bias, claiming "we extended our analyses by classifying time-varying exposure of metformin, sulfonylureas and insulin therapy" (16). The reported an adjusted HR of 0.80 (95% CI 0.65–0.98; *P* = 0.03) for metformin use relative to sulfonylurea monotherapy and the corresponding rates of cancer mortality (6.3 vs. 9.7 per 1,000 person-years) remained identical to those reported previously, which were subject to immortal time bias. This perfect equality of rates is numerically impossible and thus casts doubt on whether the time-varying statistical model was applied correctly to metformin exposure to properly reclassify the misclassified exposure in this second publication.

Immortal time bias also was evident in a study using Taiwanese National Health Insurance data in 2000 (18). It included 12,005 patients with type 2 diabetes who received at least two prescriptions of metformin during 2000–2007; these patients were compared with 4,597 patients who did not use metformin but had other OHAs and 8,643 who did not use any OHA during this period. Cancer events occurring 1 year after entry into the cohort until the end of 2007 were identified. Using the Cox proportional hazards model to adjust for covariates, the use of metformin was associated with lower incidence of cancer with HRs of any cancer of 0.12 (95% CI 0.08–0.19), of colorectal cancer (0.36 \[0.13–0.98\]), liver cancer (0.06 \[0.02–0.16\]), and pancreatic cancer (0.15 \[0.03–0.79\]).

Immortal time bias is introduced in this study from the definition of metformin exposure. Indeed, the 12,005 subjects defined as exposed to metformin during 2000–2007 had to have at least two prescriptions for metformin during this period. The bias is introduced because the time between the first and second metformin prescriptions during follow-up is immortal, that is, the patient must be cancer-free to have received the second metformin prescription, which could have been dispensed much later. Moreover, this person-time should be classified as unexposed until the second metformin prescription, at which point the remaining person-time should be classified as metformin-exposed. Thus, the analysis misclassified all this person-time as metformin exposure, which led to immortal time bias. In contrast, the comparison group of patients who did not use any OHA did not have such a condition of a second prescription.

Another example of this bias is the study using the Hong Kong Diabetes Registry to form a cohort of 2,658 patients diagnosed with type 2 diabetes who did not use metformin when enrolled in the study between 1996 and 2005 or in the 2.5 years before enrollment, with follow-up until 2005 (22). During a median 5.5 years of follow-up, 129 patients developed cancer. A Cox proportional hazards model was used to estimate the HR of cancer, which was adjusted for several covariates. The use of metformin during follow-up was associated with significant reductions in cancer incidence, with HRs of 0.51 (95% CI 0.31–0.82) among patients with HDL cholesterol ≥1.0 mmol/L and 0.30 (0.13–0.70) among patients with HDL cholesterol \<1.0 mmol/L.

Immortal time bias also is present in this study because none of the patients were using metformin at the time of entry into the cohort or during the 2.5 years before but were classified in the analysis as metformin users if they received a prescription for metformin during the follow-up. The bias then is introduced by classifying the time between entry into the cohort and the first metformin prescription as metformin-exposed. This person-time is immortal and should have been classified as unexposed until the start of metformin, at which point the remaining person-time should be classified as metformin-exposed. Misclassifying all this person-time as metformin-exposed leads to immortal time bias.

This bias also is present in a recent study investigating the association between metformin and the incidence of lung cancer (23). In that study, the authors used administrative databases from the Taiwanese National Health Insurance program to extract a cohort of 19,624 patients who were treated with antidiabetic agents between 2000 and 2005. In Cox proportional hazards models, the use of metformin was associated with a 45% risk reduction of lung cancer (HR 0.55 \[95% CI 0.37–0.82\]). Similar results were observed for thiazolidinediones and α-glucosidase inhibitors (0.55 \[0.32–0.94\] and 0.61 \[0.38–0.98\], respectively), whereas null results were observed for insulin and sulfonylureas (1.00 \[0.68–1.45\] and 1.27 \[0.75–2.15\], respectively). As with the studies described earlier, immortal time bias was introduced by misclassifying unexposed person-time as exposed person-time. Overall, the 19,624 patients generated 108,213 person-years of follow-up. Based on the data presented in the study, the 16,616 patients who ever used metformin generated 93,987 person-years of exposure, an average of 5.7 years per patient. However, the latter included immortal person-time (i.e., time between entry into the cohort and the first metformin prescription, during which no event could have occurred). Assuming an average 1-year delay between entry into the cohort and the first metformin prescription, a total of 16,616 immortal person-years would have been misclassified as exposed. As shown in Table 2, by correctly reclassifying this person-time, the rate of the unexposed group would become 33/(14,266 + 16,616) = 10.7 per 10,000 person-years instead of 23.2 per 10,000 person-years, whereas the rate in the metformin-exposed group would become 96/(93,987 – 16,616) = 12.4 per 10,000 person-years instead of 10.2 per 10,000 person-years, resulting in a corrected crude rate ratio of 1.16. Based on similar calculations, a delay of only 9.6 months between entry into the cohort and the first metformin prescription would have been necessary to bring the crude rate ratio to the null value. Thus, it is important to recognize that in the context of a highly prevalent exposure such as metformin, even small misclassifications of just a few months can result in greatly biased rate ratios.

Comparison between biased time-fixed and corrected time-dependent data analyses for the cohort study of metformin and lung cancer incidence (23)

![](2665tbl2)

As summarized in Table 1, immortal time bias affected nine observational studies evaluating the association between metformin and cancer incidence or mortality. Because of important misclassifications of exposure, the risk reductions observed in these studies were greatly exaggerated in favor of metformin. Furthermore, the variations in the reported risk reductions, ranging between 20 and 94% (Table 1), are a direct reflection of the different magnitude of misclassification of immortal time in the studies, leading to bias of different magnitudes.

## Time-window bias

A study design that has been used extensively is the nested case-control analysis conducted, in this first study, within a cohort of 22,621 women newly treated with OHAs between 1994 and 2005 who were identified using the U.K. General Practice Research Database (32). During follow-up, 309 women developed breast cancer and were matched to 1,153 controls selected from the cohort on age, general practice, and index date. Overall, the use of metformin was not associated with a decreased risk of breast cancer (odds ratio \[OR\] 1.03 \[95% CI 0.76–1.39\]). However, for patients who were prescribed at least 40 prescriptions, metformin was associated with a significant risk reduction (0.44 \[0.24–0.82\]).

In this study, the authors used risk-set sampling, making metformin exposure time-dependent and thus avoiding immortal time bias. However, the cases and controls were not matched on duration of follow-up or, more specifically, on duration of exposure opportunity time, which can lead to time-window bias (2). Matching on duration of follow-up ensures the same opportunity time of being exposed for cases and matched controls. In this study, the null finding for overall use of metformin likely indicates that cases and matched controls had, on average, similar follow-up times. However, it is possible that in certain subgroups, such as in those who received at least 40 prescriptions, the follow-up may have been different between cases and controls. As such, it is possible that controls had a longer follow-up period than cases and hence a greater opportunity to receive additional prescriptions. The opposite could have occurred as well, and therefore the direction of this bias highly depends on the distribution of follow-up among the randomly selected controls. Indeed, in a subsequent study using similar methods to investigate the effects of metformin on the incidence of colorectal cancer, the same authors reported an increased risk among patients who received at least 50 prescriptions (OR 1.43 \[95% CI 1.08–1.90\]) (36). This time-window bias also likely was present in other studies by the same authors who used this same approach to investigate the effect of metformin use on the risk of ovarian and pancreatic cancers (34,35).

Another example of time-window bias is a study investigating the association between antidiabetic drugs and the risk of hepatocellular carcinoma (31). This was a single-center, hospital-based, case-control study from Texas, involving 420 cases of hepatocellular carcinoma and 1,104 hospital controls identified between 2000 and 2008. The use of metformin and thiazolidinediones was associated with a decreased risk of hepatocellular carcinoma (OR 0.3 \[95% CI 0.2–0.6\] and 0.3 \[0.1–0.7\], respectively). It is interesting that cases were more likely than controls to be controlled with diet (13.1 vs. 2.3%, respectively). A priori, diet-controlled diabetes would have been expected to be associated with the lowest risk of cancer because it is used early in the natural history of the disease primarily in patients with a new diagnosed of type 2 diabetes (based on the assumption that disease severity is associated with an increased risk of cancer). However, it is surprising that diet-controlled diabetes was associated strongly with an increased risk of hepatocellular carcinoma (7.8 \[1.5–40.0\]). Whereas duration of diabetes was generally similar between the cases and controls, this finding reveals that duration of *treated* diabetes was largely differential between cases and controls. Indeed, the much lower prevalence of diet-controlled diabetes among the controls suggests that these patients had a longer treatment history than the cases. As such, on the basis of their longer treatment history, controls had a greater opportunity to use metformin and other antidiabetic drugs. This differential treatment time window between cases and controls likely led to the apparent increased risk of hepatocellular carcinoma with diet-controlled diabetes and consequently to an apparent decreased risk with metformin and thiazolidinediones.

As discussed earlier and summarized in Table 1, time-window bias can lead to spurious associations even when proper time-dependent analyses are performed. In some studies, there was an attempt to either adjust or match on duration of diabetes (28,31). Although controlling for this important variable certainly minimizes confounding by disease severity, it does not correct for the differential lengths of the treatment time windows in the cases and controls. Furthermore, it is difficult to predict the direction of the time-window bias because it depends on the distribution of treatment duration in the randomly selected controls compared with the cases. It is fortunate that this bias can easily be avoided at the design stage by properly accounting for duration of treatment in the cases and controls (2).

## Bias from time lag and latency

Some studies that have used cohorts of patients treated with antidiabetic drugs have compared the exposure time of metformin use with exposure time of other antidiabetic treatments, including sulfonylureas and insulin. Several methodological aspects have to be considered when a first-line therapy such as metformin is compared with second- or third-line therapies (Fig. 2). First, patients are unlikely to be at the same stage of diabetes, which can induce confounding by disease duration: longer duration of diabetes may be associated with higher cancer incidence, independent of age. In such a case, the proper comparison would require matching on disease duration so that the cohort would be formed as in Fig. 2 (*bottom*) rather than the time-lagged comparison suggested by Fig. 2 (*top*). Second, if the first treatment used is associated with an increased incidence of cancer, albeit after a long period of exposure, it is likely that the cancer will occur during exposure to the second-line agent, in which case attribution of the cancer to either treatment is challenging—thus the importance of considering latency time windows.

A study that was affected by confounding by disease stage is one that used The Health Information Network, a U.K. general practice database (17). In this study, the authors identified 62,809 users of OHAs or insulin between 2000 and 2007. The study population was then subdivided into four cohorts: cohort 1 included new users of metformin monotherapy; cohort 2 comprised new users of sulfonylurea monotherapy; cohort 3 included those who switched from monotherapy with metformin or sulfonylurea to a combination of both drugs; and cohort 4 included those who switched from OHAs to insulin. Incidence rates of cancer (specifically breast, pancreas, colorectal, and prostate cancers) were calculated within each of the aforementioned cohorts, and Cox proportional hazards models were used to compare cohorts 2, 3, and 4 with cohort 1 (metformin monotherapy). After a mean follow-up of 2 years, the HRs (95% CIs) were 1.4 (1.2–1.5) for sulfonylurea monotherapy, 1.1 (1.0–1.2) for the metformin-sulfonylurea combination, and 1.4 (1.3–1.6) for insulin. Among patients treated with insulin, the concomitant use of metformin was associated with a statistically significant risk reduction (0.54 \[0.43–0.66\]). However, this strong inverse association was likely due to immortal time bias because exposure to metformin was assessed at any time during follow-up and thus included person-time during which patients were not yet exposed to this therapy (Table 1). Furthermore, as noted by others, the survival curves of the different cohorts presented by the authors diverged as early as during the first year of treatment, thus arguing against a causal effect, which is biologically inconsistent with the long latencies of cancers (38). Aside from reverse causality and confounding, which are certainly important considerations, such early divergence in the survival curves can also be explained by two other factors. First, comparing the different cohorts, which consisted of second- and third-line treatments, with metformin monotherapy, a first-line treatment, inherently introduces biases related to disease duration and progression that may not be dealt with adequately during analysis. Second, the consideration of cancers diagnosed early after entry into the cohort is problematic, especially for patients with a history of OHA use (i.e., cohorts 3 and 4), because it ignores the fact that previous exposure might have affected future cancer risk. Indeed, patients may remain at risk for an extended period of time long after discontinuation of treatment. For example, former smokers with a history of smoking one pack per day for a period of 1 year have a nearly threefold increased risk of lung cancer after 30 years of cessation (39). One approach to minimize this bias is to apply time-lag periods to exclude cancers diagnosed within a specified time period after entry into the cohort. This method works reasonably well, especially if there is clustering of cancers early during the treatment, as was observed in this study (17).

A study that introduced a variation of bias due to time-lagging of comparison cohorts was that based on the Diabetes Audit and Research in Tayside Study cohort of patients diagnosed with type 2 diabetes in Tayside, Scotland (37). All 4,085 eligible new users of metformin between 1994 and 2003 were matched individually with 4,085 nonusers on year of diabetes diagnosis; each pair was assigned the date of entry into the cohort as the date of the first metformin prescription of the user. Of the subjects who were cancer free at cohort entry, 771 developed cancer during follow-up. Cancer was diagnosed in 7.3% of the metformin users compared with 11.6% of nonusers. A Cox proportional hazards model was used to estimate the HR of cancer, adjusted for several covariates. The adjusted HR of cancer associated with metformin use was 0.63 (95% CI 0.53–0.75), suggesting a strong protective effect of metformin.

Although this study avoided biases associated with prevalent exposures by using a new-user cohort of patients treated with metformin, this approach was not used for the comparison group, which was not based on a specific comparison drug. Because the comparison metformin nonuser does not have a prescription date to define time zero, it was assigned the date of the first metformin prescription of its matched user, a date called the index date. In choosing among potential nonusers for a given user, a variation of bias from time lags was introduced. Indeed, if the potential matched nonuser had a diagnosis of cancer or had died prior to the index date, it was discarded. However, it "was potentially available for a different metformin user…this process was repeated until suitable comparators were identified."

Nonusers should be alive and cancer free at entry into the cohort just like the matched users, but the net effect of selecting the nonusers with replacement is twofold. First, the final nonuser group had zero subjects excluded because of cancer before the index date, in contrast with the 239 excluded from the metformin group. Second, the nonusers who should have been excluded (and not reused) because of a previous cancer were included as nonusers matched to other users. As a result, cancers that should have been an exclusion criterion were transferred to the follow-up period after the index date and counted as outcome events, thus artificially increasing the rate of cancer in the nonuser group. This phenomenon is noticeable in the Kaplan-Meier curves that suggest a clustering of cancers in the first 2 years of follow-up of the nonuser group. Thus, by forming the nonuser group, the authors artificially induced a cohort that was immortal and cancer free before the index date but had more deaths and cancers after entry into the cohort. As a result of this transfer, the nonuser group had 474 cancers diagnosed during follow-up compared with 297 in the metformin group. If the exclusion of 239 cancers before the first metformin prescription is applied to the nonuser group, the 474 would be reduced to 177.

# STUDIES OF CANCER TREATMENT

A number of studies investigated the effects of metformin among patients with cancer. Several of these studies reported significant risk reductions for all-cause mortality, cancer-specific mortality, and cancer progression, ranging between 34 and 62% (24–27). All of the studies reporting these important risk reductions suffered from immortal time bias (summarized in Table 1). We describe below selected studies to illustrate this bias in the setting of cancer treatment.

In the first study, the authors reviewed the records of 233 patients diagnosed with prostate cancer between 1999 and 2008 at a medical institution in Texas (24). They evaluated whether antidiabetic treatments were associated with all-cause mortality in this patient population. Cox proportional hazards models were constructed, with ever-use of insulins and OHAs entered as independent variables, and adjusted for potential confounders. Their results indicate that metformin (HR 0.55 \[95% CI 0.32–0.96\]) and thiazolidinediones (0.45 \[0.21–0.97\]) were associated with a significant decrease in all-cause mortality. No statistically significant associations were observed with the other antidiabetic agents, although few patients were exposed in this small dataset. Misclassifying as exposed the time between cohort entry and first prescription of metformin or a thiazolidinedione introduced immortal time bias, thus greatly exaggerating the benefits of these drugs on all-cause mortality. That exposure was defined as any time during the 10-year follow-up period contributed to compound the effect of this misclassification. As explained earlier, the appropriate analysis would have treated exposures to the different antidiabetic agents as time-dependent variables.

The authors of another study identified 595 patients who had been diagnosed with both colorectal cancer and diabetes mellitus and compared the 258 taking metformin any time during follow-up with the 337 nonusers (25). After a median follow-up of 41 months, the adjusted HR of all-cause mortality with metformin use was 0.66 (95% CI 0.45–0.98), whereas for colorectal cancer, mortality was also 0.66 (0.48–0.92). Here again, these mortality reductions are the result of immortal time bias caused by misclassifying as exposed to metformin the time between cohort entry and first prescription, when in fact it is unexposed.

Immortal time bias also was present in a cohort study of 1,983 consecutive patients with HER2+ breast cancer treated between 1 January 1998 and 30 September 2010 (26). All-cause and cancer-specific deaths were identified. Among the patients with diabetes, the HR of all-cause death associated with metformin was 0.52 (95% CI 0.28–0.97), whereas for breast cancer–specific mortality it was 0.47 (0.24–0.90). These mortality reductions again are the result of immortal time bias caused by misclassifying as exposed the time between cancer diagnosis and first prescription of metformin given during follow-up, when in fact it is unexposed to metformin.

# CONCLUSIONS

We have shown that a large number of observational studies reporting significant reductions in the incidence of or mortality from cancer associated with metformin use are afflicted with time-related biases such as immortal time bias, time-window bias, and time-lag bias. By not classifying metformin exposure during follow-up or measuring metformin exposure over different time intervals properly, the resulting analyses produced apparent reductions in risk that were created artificially by the misclassification of metformin exposure. These biases, regrettably common in pharmacoepidemiology, have been described extensively in different therapeutic areas (1,2,40–42) but do not seem to have sufficiently penetrated the fields of diabetes and cancer. Although two of the studies recognized the possibility of immortal time bias, the technique of data analysis required to address it was clearly not appropriate (16,18).

Other studies have been conducted with varying results; some have found no association between metformin use and cancer incidence or prognosis (43–48), others have found lower rates of cancer with metformin (49–57), and one study found an increased risk (58). Although these studies were designed and analyzed with no obvious time-related biases, it is of course possible that other epidemiological biases such as selection, information, or confounding could have affected them. This article focused specifically on time-related biases, where metformin was associated with strong risk reductions, ranging from relative risks of 0.80 (20% risk reduction) to 0.06 (94% risk reduction), likely due to flaws in their designs and methods of analysis.

Three recent studies that specifically used the time-dependent techniques needed to avoid both immortal time and time-window biases found no association between metformin use and cancer incidence (59–61). The first study used the U.K. General Practice Research Database and found a rate ratio of prostate cancer incidence of 1.23 (95% CI 0.99–1.52) with metformin use. With more than 36 prescriptions the rate ratio was significantly elevated at 1.40 (1.03–1.89). The second study used the Kaiser Permanente database and found no effect of metformin on the incidence of the 10 different cancers studied, with HRs ranging between 0.8 (0.6–1.1) for melanoma to 1.3 (1.0–1.6) for kidney/renal and pelvic cancers. Finally, no effect of metformin was found on lung cancer incidence (61).

It is also important to recognize that most observational studies focused on the incidence of cancer as it relates to the effects of metformin on carcinogenesis, which is distinct from its effects on proliferation of established tumors. Nevertheless, large RCTs using patients with established tumors currently are being planned or already are in progress. One such study is the MA.32 being conducted by the National Cancer Institute of Canada, a multicenter phase III RCT that includes almost 3,600 women with early breast cancer and is comparing metformin to placebo as adjuvant therapy on the primary end point of invasive, disease-free survival over a 5-year follow-up (12,13). The authors claim that "we believe the science underlying such a trial is strong, the novelty of the intervention is high, and the potential for benefit is large" (12). Although it is possible that the in vitro science is strong and metformin may lead to positive results, we have shown that the pharmacoepidemiological evidence from observational studies is weak at best.

In conclusion, time-related biases are common in the field of diabetes and can be avoided using appropriate study designs and methods of analysis. In the case of the association between metformin and cancer, we believe that methodologically inaccurate studies, even if their results are replicated in different settings, should not be the driving force behind long and expensive trials. At this time, it may be important to question and reassess the scientific evidence of the potential beneficial effects of metformin on cancer. Although the in vitro science seems to be strong, certainly all observational studies identified as potentially biased would need to be redone properly to correct for the bias. Moreover, new observational studies using more rigorous designs and analyses that avoid these biases should urgently be undertaken before more randomized trials are initiated in vain.

## Acknowledgments

S.S. is the recipient of the James McGill Chair, and L.A. is the recipient of a Chercheur-Boursier Award from the Fonds de la recherche en santé du Québec.

No potential conflicts of interest relevant to this article were reported.

# References


abstract: # Introduction
  .
  Angiotensin-converting enzyme (ACE) 2, a homolog of ACE, converts angiotensin (Ang) II into Ang(1-7), and the vasoprotective effects of Ang(1-7) have been documented. We explored the hypothesis that serum autoantibodies to ACE2 predispose patients with connective tissue diseases to constrictive vasculopathy, pulmonary arterial hypertension (PAH), or persistent digital ischemia.
  .
  # Methods
  .
  Serum was examined from 42 patients with systemic lupus erythematosus (SLE), scleroderma, or mixed connective tissue disease. Eighteen vasculopathy patients with PAH (five cases) and/or persistent digital ischemia (16 cases) were compared with 24 patients without these vasculopathies (control patients) for serum reactivity to purified recombinant human ACE2, using an ELISA.
  .
  # Results
  .
  The sera from 17 of the 18 (94%) vasculopathy patients had ELISA scores above the baseline level determined using control sera from 28 healthy subjects, and the mean ELISA score in the vasculopathy patients was significantly higher than that in the control patients (*P* \< 0.0005). The relative activity of serum ACE2, which was defined using a reference serum, correlated inversely with the ELISA scores for serum anti-ACE2 antibodies in the 18 vasculopathy patients (*R*^2^ = 0.6872). The IgG fraction from vasculopathy patients, but not from healthy subjects, inhibited ACE2 activities *in vitro*. Consistent with this, immunosuppressive therapy given to one SLE patient with digital necrosis markedly decreased the anti-ACE2 antibody titer and restored serum ACE2 activity.
  .
  # Conclusions
  .
  Autoantibodies to ACE2 may be associated with constrictive vasculopathies.
author: Yuko Takahashi; Shiori Haga; Yukihito Ishizaka; Akio Mimori
date: 2010
institute: 1Division of Rheumatic Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan; 2Department of Intractable Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
references:
title: Autoantibodies to angiotensin-converting enzyme 2 in patients with connective tissue diseases

See related editorial by Chappell, <http://arthritis-research.com/content/12/3/128>

# Introduction

Angiotensin-converting enzyme (ACE) 2, a homolog of ACE, is a carboxypeptidase that degrades angiotensin (Ang) II to Ang(1-7) \[1\]. Ang(1-7) has vasodilating, antiproliferative, and antithrombotic properties that antagonize the action of Ang II and play vasoprotective roles \[2-4\]. Recent studies have demonstrated the therapeutic effects of ACE2 activation by a synthetic molecule \[5\] or of *ACE2* gene transfer \[6\] in experimental pulmonary hypertension models.

Pulmonary arterial hypertension (PAH), a vasculopathy of unknown etiology, is a serious complication of connective tissue disease (CTD) \[7\]. One clinical study found reduced metabolism of ACE synthetic substrate in the pulmonary vascular bed of PAH-CTD patients, but not in primary PAH patients \[8\]. Persistent digital ischemia, which manifests as skin ulcers or necrotic lesions, is another intractable vasculopathy of CTD, and is strongly associated with Raynaud's phenomenon. A correlation between Raynaud's phenomenon and elevated systolic pulmonary arterial pressure has been reported in patients with systemic lupus erythematosus (SLE) \[9\]. PAH or persistent digital ischemia is less frequent than Raynaud's phenomenon, and these three vascular abnormalities are involved in CTD patients across different disease entities, including SLE, systemic sclerosis (SSc), and mixed connective tissue disease (MCTD).

Our preliminary examination suggested the presence of novel autoantibodies to ACE2 in the sera of two patients: a patient with SLE suffering from severe digital necrosis, and a patient with SSc accompanied by lethal PAH. Furthermore, the sera of the two patients lacked ACE2 activity. These findings prompted us to conduct the present study in order to explore the hypothesis that serum autoantibodies to ACE2 predispose patients with CTD to constrictive vasculopathies; that is, PAH and persistent digital ischemia.

# Materials and methods

## Study design

As many patients as possible among those with CTD and PAH or persistent digital ischemia (vasculopathy patients) in our hospital at time of the study were enrolled. Sera from these patients were studied in comparison with those from CTD patients without vasculopathy or from healthy subjects. The ethics committee of our hospital approved this study, and written informed consent was obtained from all patients and control subjects.

## Serum sampling

Fresh serum was obtained from all of the patients and normal subjects for the present study. Each serum sample was aliquoted to avoid repeated thawing and was stocked at -20°C until assayed.

## Diagnosis of connective tissue disease

Forty-two patients with SLE, SSc, or MCTD were studied. SLE was diagnosed according to the classification criteria of the American College of Rheumatology \[10\]. Patients with SSc met the classification criteria for the diffuse (n = 3) or limited (n = 6) form of SSc, as described in the literature \[11\]. Patients with MCTD met the criteria for MCTD from Kasukawa and colleagues \[12\] and the original definition of MCTD by Sharp and colleagues \[13\].

## Diagnosis of pulmonary arterial hypertension

PAH had been diagnosed in five patients with SSc, based on dyspnea on exertion, elevated plasma brain natriuretic peptide levels \>100 pg/ml, right ventricular outflow and peak tricuspid regurgitant pressure gradient exceeding 30 mmHg on echocardiography, exclusion of pulmonary thromboembolism by high-resolution computed tomography or pulmonary scintigraphy, and no deteriorated lung fibrosis that could cause pulmonary hypertension.

## Diagnosis of persistent digital ischemia

Each of the 16 patients in this category had persistent cyanotic lesions on the digits, present for more than 2 years at the time of the present study, and a history of or existing digital ulcers or necrosis. The radial artery pulse in each patient was normal, and atherosclerotic ischemia was excluded clinically. Three of the 16 patients also had PAH.

## Control patients

Serum samples from 24 CTD patients who had neither PAH nor persistent digital ischemia were collected randomly (that is, independent of the activity of their CTD) on the ward or in the outpatient clinic. These samples served as disease controls.

## Normal control subjects

Serum samples were obtained from 28 healthy volunteers (23 females and five males) who were free from any active or chronic diseases, including hypertension. Before bleeding, each subject was confirmed to have normal blood pressure in the arms. The mean age of the 28 subjects was 32.0 ± 9.5 years.

## Detection of anti-ACE2 antibodies in patient serum

We established an ELISA using purified recombinant human ACE2. A plasmid DNA-encoding human ACE2 cDNA, which was kindly gifted from Dr Hyeryun Choe (Harvard Medical School, Cambridge, MA, USA), was introduced into 293 free-style cells, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Culture supernatant was harvested on day 2 after transfection, dialyzed against 25 mM Tris-Cl (pH 8.5), and applied onto a DEAE column. ACE2 was eluted with 250 mM NaCl, and was dialyzed against for further use. About 12.5 μg/ml recombinant human ACE2 were first coated overnight onto a 96-well plate with bicarbonate buffer (pH 9.6) at 4°C. The wells were then treated with a blocking buffer composed of 5% BSA in PBS and washed with buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20. The sera of the patients and normal healthy volunteers were added to the plate and incubated for 1 hour at room temperature. Bound ACE2 antibodies were detected using horseradish peroxidase-conjugated anti-human IgG antibody. The optical density at 450 nm was measured after a 30-minute incubation with SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA). All samples were analyzed in triplicate, and the values were normalized with the positive control.

## Measurement of serum ACE2 activity

Zinc-dependent ACE2 enzymatic activity was measured according to the methods described previously \[14,15\]. Briefly, 20 μM (7-methoxycoumarin-4-yl) acetyl-APK-(2,4-dinitrophenyl)-OH (amino acids depicted by single letters) (AnaSpec Inc., Fremont, CA, USA) - a fluorogenic substrate \[14\] - was incubated with 1 μl test serum. Fluorescence was monitored using Safire2 (TECAN, Männedorf, Switzerland) at the excitation and emission wavelengths of 320 nm and 450 nm, respectively.

Preliminary experiments revealed that the relative fluorescence unit (RFU) value was not increased when the substrate was simply incubated without the enzyme (data not shown). Among 20 healthy humans, two samples showed significantly high autofluorescence - in these cases, the RFU values decreased during the incubation even with the substrate. We excluded these samples because it was not possible to evaluate the ACE2 enzyme activity.

The RFU values obtained by the APK substrate were almost completely inhibited by an ACE2 inhibitor, DX600 (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) (Additional file 1). Additionally, the RFU values were statistically significant at both 60 and 70 minutes of the incubation (Additional file 2). Based on these observations, we used the RFU values at 70 minutes without subtracting the residual RFU counts resistant to DX600 \[15\]. The relative ACE2 activity (%) in each sample was further calculated based on the level of ACE2 activity in a mixture of serum samples from 28 healthy subjects (a reference serum). The assay was performed in triplicate and the mean values of three independent measurements of the ACE2 ELISA and ACE2 activity assay were used for the statistical analysis by Student's *t* test.

## Detection of serum ACE2 protein

An anti-human ACE2 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA) was used for immunoprecipitation. Serum was first treated for 15 minutes with 100 mM MgCl~2~, 70 units DNase I (Takara Bio Inc., Shiga, Japan) and 500 μg/ml Ribonuclease A (Sigma, St Louis, MO, USA) at room temperature, and was then incubated with anti-human ACE2 antibody at 4°C for 2 hours. After incubation, the immune complexes were recovered using Protein G Sepharose™ 4 Fast Flow (GE Healthcare, Amersham, Buckinghamshire, UK) and subjected to SDS-PAGE, followed by western blot analysis. To detect ACE2 proteins, an anti-human ACE2 mouse monoclonal antibody was used (R&D Systems).

## Purification of IgG from serum

Five microliters of serum were incubated with Protein G Sepharose™ 4 Fast Flow in 20 mM sodium phosphate buffer (pH 7.0) for 2 hours at 4°C. The immune complexes were then washed and the IgG-bound beads eluted with 0.1 M glycine-HCl (pH 2.7). The recovered IgG was immediately neutralized with 1 M Tris-HCl (pH 8.0). IgG was detected by SDS-PAGE followed by Coomassie Brilliant Blue staining. IgG fractions were prepared from three vasculopathy patients and three healthy subjects. The effect of each IgG on the activity of recombinant human ACE2 (standard rACE2; R&D Systems) was examined as described.

# Results

## Disease status of patients at the time of the present study

The demographics of patients with CTD and PAH or persistent digital ischemia (vasculopathy patients, n = 18) and of the control CTD patients (n = 24) are presented in Table 1. The control patients had neither manifestations nor a history of these vasculopathies.

Patient demographics

| Parameter | Vasculopathy patients | Control patients |
|----|----|----|
| Number of patients | 18 (17 females, 1 male) | 24 (21 females, 3 males) |
| Age (years) | 50.5 ± 14.3 | 46.8 ± 17.8 |
| Disease entities | SLE (4), SSc (6), MCTD (8) | SLE (21), SSc (2), MCTD (1) |
| Constrictive vasculopathies | PAH (5); all patients had SSc^a^; persistent digital ischemia (16) | None |

Data presented as n or mean ± standard deviation. PAH, pulmonary arterial hypertension; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; SSc, systemic sclerosis. ^a^Three patients also had digital ulcers; of these, one patient had a history of scleroderma renal crisis.

### Vasculopathy patients

Of the 18 vasculopathy patients, three (Patients 1, 5, and 12) were hospitalized for treatment of an exacerbation of their CTD. Patient 1, with SLE, had fever, pancytopenia, and progressing digital necrosis of the fingers and toes. Patient 5, with the diffuse form of SSc, was referred to our hospital and admitted because of heart failure caused by severe PAH of recent origin. Despite high-dose steroid therapy for PAH and conventional therapy for heart failure, she died of sudden cardiac arrest. Patient 12, with SLE, had fever, pancytopenia, diffuse erythema, and painful digital ischemia accompanied by chilblain lupus.

The remaining 15 outpatients (all had persistent digital ischemia and four also had PAH) were being treated with prostacyclins or calcium antagonists without apparent improvement, and all of the patients with digital ischemia had suffered from digital ulcers or digital necrosis within 2 years of the present study. In the five patients with PAH, the mean ± standard deviation peak tricuspid regurgitant pressure gradient was 51 ± 13 mmHg (range, 39 to 70 mmHg; median, 50 mmHg) on echocardiography within 6 months of the present study, and the mean plasma brain natriuretic peptide level was 639 ± 623 pg/ml (range, 107 to 1,312 pg/ml; median, 568 pg/ml) at the time of this study. Systemic hypertension was observed in four SLE patients and in one SSc patient. Three of the patients had been treated with a calcium antagonist or ACE inhibitor for years, and hypertension developed in the remaining two patients (Patients 1 and 12) during steroid therapy at the time of study.

### Control connective tissue disease patients

Of these 24 patients, 11 were hospitalized: nine patients for exacerbated SLE (six with lupus nephritis, two with neuropsychiatric lupus, and one with thrombocytopenia), one SLE patient for aseptic bone necrosis, and one SSc patient for accidental cholecystitis. The remaining 13 outpatients had stable disease on a maintenance dose of steroid therapy. Recent Raynaud's phenomena were observed in two SLE patients, two SSc patients, and one MCTD patient. Systemic hypertension was observed in one SLE patient, who had been treated with an Ang II receptor blocker.

## ELISA for serum anti-ACE2 antibodies

All of the patients had received a maintenance dose of steroid therapy by the time of blood sampling. Serum samples were obtained from inpatients just before starting additional immunosuppressive therapy for a disease flare-up, and from outpatients. Sera from 17 of the 18 (94%) vasculopathy patients showed reactivity to ACE2 with ELISA values above the baseline level value, which was determined as the mean ELISA score in the 28 normal control subjects. The mean ELISA score was significantly higher in the vasculopathy patients than in the control patients (Figure 1). These results suggest that anti-ACE2 antibodies in the serum are associated with constrictive vasculopathies, PAH, or persistent digital ischemia.

## Inhibition of ACE2 activity by IgG purified from patient serum

IgG fractions from the sera of three healthy subjects and three vasculopathy patients (Patients 1, 5, and 6) were prepared (Figure 2a). The activity of standard rACE2 was measured in 5 μg aliquots. The IgG fractions from the vasculopathy patients, but not from the healthy subjects, suppressed the rACE2 enzyme activity significantly compared with that in the absence of IgG (Figure 2b).

## Serum ACE2 activity in vasculopathy patients, control patients, and healthy subjects

First, the RFU value of the ACE2 enzyme activity, which was blocked by DX600 (Additional file 1), was determined in 18 patients with vasculopathy, in 16 control patients without vasculopathy, and in 26 healthy subjects. The relative rate of serum ACE2 enzyme activity was then compared with a reference mixture composed of sera from healthy subjects. Serum ACE2 activity could not be measured in one control patient and two healthy subjects due to high fluorescence at the start of incubation, and sera from the remaining seven control patients were not available for these experiments. The mean ± standard deviation relative ACE2 activity was significantly lower in the vasculopathy patients (64.8 ± 16.4, n = 18) compared with that in the healthy subjects (86.6 ± 32.2, n = 26, *P* = 0.0055) and control patients (165.1 ± 99.1, n = 16, *P* = 0.0010).

To clarify the correlation between ACE2 activity and the presence of autoantibodies to ACE2, the ELISA scores for serum anti-ACE2 antibodies and serum ACE2 activity were plotted (Figure 3). Our data indicate that the titers of anti-ACE2 antibodies were inversely proportional to ACE2 peptidase activity in the vasculopathy patients (Figure 3c). In contrast, no correlation was observed in the healthy subjects or control patients, suggesting that the low serum ACE2 activities in most of the vasculopathy patients were due to the presence of anti-ACE2 antibodies. To assess ACE2 expression in the vasculopathy patients, we tested for ACE2 by immunoprecipitation followed by immunoblotting. As shown in Figure 3d, the ACE2 levels in Patients 1 and 5, who were deficient in ACE2 activity (Figure 3c), were comparable with those in three healthy subjects, clearly indicating that the decreased ACE2 activity in the vasculopathy patients was due to a functional deficiency. The relative ACE2 protein levels in the patients are shown in Additional file 3. The signal intensities of ACE2 protein and the IgG heavy chain shown in Figure 3d were measured and normalized.

## Recovery of serum ACE2 activity after therapy in one patient

The exacerbated SLE and progressive necrosis of the fingers and toes in Patient 1, a 25-year-old female, were treated with intravenous methylprednisolone at a dose of 500 mg/day for 3 days, followed by 30 mg/day oral prednisolone combined with three sessions of double-filtration plasmapheresis and conventional therapy, including prostanoids, stellate ganglion anesthesia, and local care. Her cutaneous lesions healed completely before discharge, without amputation of any digits. Serum tests 2 months after the start of therapeutic intervention showed that the ELISA score for anti-ACE2 antibodies was reduced markedly (*P* \< 0.05) (Figure 4a) and the ACE2 activity had recovered significantly (*P* \< 0.05) (Figure 4b), compared with the values before therapy. An ELISA for anti-ACE2 antibodies in the waste double-filtration plasmapheresis fluid was negative, and thus we could not evaluate whether double-filtration plasmapheresis had removed these antibodies from the patient's blood. The anti-ACE2 antibodies were probably decreased as a result of immunosuppressive therapy using high-dose pulse steroids.

The other two vasculopathy inpatients (Patients 5 and 12) also received immunosuppressive therapy during the present study and had high ELISA scores for anti-ACE2 antibodies before therapy. The serum of Patient 5 was not studied after therapy, because she died suddenly. For Patient 12, the ELISA scores before therapy and after 2 months of therapy with 30 mg/day prednisolone were 0.68 ± 0.02 and 0.36 ± 0.01, respectively. These results were not significantly different, and the titer tended to decrease after therapy. The relative rates (%) of serum ACE2 activity in this patient before and after therapy were 52.2 ± 19.1 and 63.1 ± 4.8 (*P* = NS), based on the level of activity detected in the reference sample, respectively.

# Discussion

Persistent digital ischemia and clinically evident PAH are intractable and relatively infrequent complications of SLE, SSc, and MCTD - although latent forms of vasculopathies, including sporadic abnormalities such as Raynaud's phenomena, may be more prevalent in CTD patients. Immunosuppressive therapy can improve PAH in some patients with SLE, SSc, or MCTD \[16\], and in most SLE patients \[17\] via an unknown therapeutic mechanism. The treatment of digital ischemia remains an unsolved clinical problem for physicians. Our study suggests, for the first time, that autoantibodies to ACE2 are present in the serum of patients with persistent constrictive vasculopathies. Furthermore, the elevated ELISA scores for anti-ACE2 antibodies correlated with reduced ACE2 activity. The serum autoantibodies to ACE2 may therefore inhibit ACE2 activity, and the inhibition may be reversible after immunosuppressive therapy, based on the data from Patient 1. There were small numbers of patients with systemic hypertension in both the vasculopathy and control patient groups. We could not determine whether their hypertension was essential hypertension, or was caused by steroid therapy or ACE2 inhibition.

The inhibition of ACE2 by autoantibodies may result in reduced physiological levels of the vasoprotective agent Ang(1-7) in the local vascular milieu, which may induce vasculopathies in patients with underlying disease such as CTD. Vasodilating drugs - including prostanoids, endothelin-receptor antagonists, and phosphodiesterase type-5 inhibitors - have been used widely to treat PAH and have been partially effective for some PAH patients \[18\]. No effective drugs have been found for persistent digital ischemia \[19\]. Our study suggests that ACE2 activation or administration in experimental PAH \[5,6\] may be applicable as a therapy for PAH or persistent digital ischemia in patients with CTD.

# Conclusions

Anti-ACE2 autoantibodies may be associated with constrictive vasculopathy in patients with connective tissue disease.

# Abbreviations

ACE: angiotensin-converting enzyme; Ang: angiotensin; BSA: bovine serum albumin; CTD: connective tissue disease; ELISA: enzyme-linked immunosorbent assay; MCTD: mixed connective tissue disease; PAH: pulmonary arterial hypertension; PBS: phosphate-buffered saline; rACE2: recombinant human angiotensin-converting enzyme 2; RFU: relative fluorescence unit; SLE: systemic lupus erythematosus; SSc: systemic sclerosis.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

YT and SH equally contributed to this study. YT performed the ELISA, clinical practice, and wrote the paper. SH prepared a recombinant ACE2 protein, carried out the assays for ACE2 activities, and wrote the methods. YI designed the experiments. AM designed the research and completed the paper.

# Supplementary Material

###### Additional file 1

**Inhibition of the enzyme activity by an ACE2 inhibitor**. By preincubation with DX600 for 30 minutes, the enzyme activity was almost completely blocked (*P* \< 0.01). H, healthy volunteer; P, patients with vasculopathy; CP, control patients without vasculopathy. The activity was also blocked by the addition of ethylenediamine tetraacetic acid (EDTA) (data not shown), indicating that the activity would depend on the presence of zinc ion.

Click here for file

###### Additional file 2

**Optimization of the measurement analysis of ACE2 activity**. ACE2 activity was measured chronologically with the fluorogenic substrate and plotted for 90 min. The relative fluorescence unit (RFU) values increased within 70 minutes and then declined. Each plot is a representative result of three independent experiments. In each experiment, a rACE2 (a standard) and a reference serum (a mixture of sera from 28 healthy subjects) were assayed simultaneously with sample sera from healthy subjects, control patients, or vasculopathy patients. The difference of the ACE2 enzyme activity between healthy subjects and vasculopathy patients was statistically significant (*P* \< 0.01) both at 60 and 70 minutes of the incubation.

Click here for file

###### Additional file 3

**Relative intensity of the ACE2 protein levels in patients**. The signal intensities of ACE2 protein and the IgG heavy chain shown in Figure 3d were measured and normalized. Each relative intensity was standardized with that of sample H1.

Click here for file

## Acknowledgements

The authors thank Dr Hyeryun Choe (Harvard Medical School, Cambridge, MA, USA) for a plasmid DNA encoding human ACE2 cDNA. The present work was supported by Grants-in-Aid for Research on Intractable Diseases from the Ministry of Health, Labor, and Welfare of Japan.


author: Hans Erik Bøtker; Niels MøllerCorresponding author: Niels Møller, <email@example.com>.
date: 2013-08
references:
title: ON NO—The Continuing Story of Nitric Oxide, Diabetes, and Cardiovascular Disease

Nitric oxide (NO) is a simple chemical compound—1 nitrogen and 1 oxygen atom coupled together—with complex biological actions (1,2). A singularly prominent feature of NO is its ability to cause vasodilation, a quality that is used pharmacologically when treating ischemic heart disease with NO precursors such as nitroglycerin.

In 1980, Furchgott and Zawadzki (3) showed that vascular relaxation induced by acetylcholine was dependent on the presence of endothelium and provided evidence for the release of a volatile humoral factor. This substance, later called endothelium-derived relaxing factor and now recognized as NO (4), is a significant component of the insulin-signaling cascade (Fig. 1), executing microvascular vasodilation stimulated by local NO production from the vascular endothelium (5). Vasodilation has the potential to decrease systemic blood pressure and increase local tissue blood flow in tissues such as muscle. The combination of decreased blood pressure and increased tissue blood flow, together with specific beneficial endothelial effects, may serve to prevent hypertension, cardiovascular disease, and insulin resistance (5,6). However, these effects remain exquisitely sensitive to impaired insulin signaling within the endogenous cardiovascular NO system and appear to be compromised in the presence of insulin resistance (7). So there is evidence that impaired NO-dependent vasodilation causes hypertension and insulin resistance and vice versa: that insulin resistance, such as that observed in diabetes, the metabolic syndrome, and hypertension, impairs NO-dependent vasodilation. The existence of this vicious cycle is supported by epidemiological data from the Framingham Heart Study and other studies showing that hypertension, diabetes, and cardiovascular disease cosegregate (8,9).

In the vasculature, NO is synthesized from the guanidine group of the amino acid arginine under critical control of the enzyme endothelial NO synthase (eNOS). eNOS activity is stimulated by insulin (and estrogen), exercise, and caloric restriction (10,11), whereas gluco/lipotoxicity, inflammation, and proinflammatory cytokines, together with specific arginine metabolites, ADMA (asymmetric dimethylarginine), LMMA (l-monomethylarginine), and SDMA (symmetric dimethylarginine), inhibit eNOS (10,12,13). It is important to emphasize that insulin has dual actions (11): it acts after binding to its receptor and activates phosphatidylinositol 3-kinase (leading to eNOS stimulation, NO production, and vasodilation) and mitogen-activated protein kinase (leading to endothelin-1 stimulation and vasoconstriction/cell adhesion), as illustrated in Fig. 1. Given the complex nature of the regulation of vascular tone and endothelial function, it is of major importance to define and describe the underlying mechanisms leading to vascular dysfunction and insulin resistance. From a clinical point of view, there is a specific need for experimental human studies assessing NO synthesis and clearance rates and whether abnormalities of NO function relate to altered kinetics and impaired insulin stimulation.

In this issue of *Diabetes*, Tessari et al. (14) report basal and insulin-stimulated synthesis rates for mononitrogen oxides (NO and NO~2~ – NOx) in 26 subjects covering a wide range of age, BMI, cholesterol, glucose tolerance, and renal function. In these experiments, the investigators use a precursor product, isotope dilution technique. Because arginine is the precursor for NO (Fig. 1), arginine labeled with nitrogen-15, a stable isotope, is infused, and the percentage of labeled arginine (precursor) and its product (NO) is measured by mass spectrometry. Once the whole system is at steady state, synthesis/appearance rates for arginine and NO can be calculated depending on the "intensity" of labeling. Compared with normal subjects, the new data showed that NO synthesis was lower in the elderly and in people with type 2 diabetes and generally increased after insulin stimulation. Regression analysis using data for all subjects showed that NOx synthesis was inversely correlated with arginine metabolites (ADMA, SDMA) and age, but not with insulin sensitivity. The authors conclude that whole-body NOx production is decreased in aging and type 2 diabetes and that arginine metabolites, not insulin resistance, appear to be negative regulators of in vivo NOx production.

These are timely and pertinent data from a well-conducted human study, and the findings not only expand our understanding of the field but also suggest that insulin resistance and NO dysfunction may not be as intimately linked as hitherto believed. A particular strength of the study is that it combines a complicated clinical setup in a relative large number of subjects with a state-of-the-art kinetic tracer technique.

A recent study, using a saliva oral nitrate test and an intravenous glucose tolerance test, reported a correlation between insulin sensitivity and NO synthesis (15), whereas the current study, using steady state isotope dilution and clamp techniques, fails to make this connection. Insulin sensitivity is expressed by a glucose clamp–derived M (or glucose infusion rate) value, which predominantly reflects glucose uptake in muscle (16). NO synthesis is measured systemically, leaving the contributing tissues unidentified. Hence, the new results do not necessarily reflect conditions in muscle. Furthermore, the design of the study is quite complex, with many different subgroups and mixed pathologic features. It is therefore possible that the heterogeneity of the study sample may have underpowered the study. Whether clearance/disappearance is altered in insulin-resistant states is not known, and it is possible that NO-dependent alterations in blood flow might contribute to a feedback loop in which NO removal from its target tissues increases with increasing vasodilation and blood flow.

The clinical implication of the current study relates to the effect of insulin resistance on endothelial dysfunction. Although Tessari et al. (14) did not measure endothelial function or any balance between insulin-regulated NO-dependent vasodilator actions versus endothelin-1–dependent vasoconstrictor actions, the data support the idea that insulin resistance may only partly explain the impairment of endothelial function with increasing age, obesity, and type 2 diabetes. Several studies using multivariate analyses that adjust for other potential modulators of endothelial function have shown that insulin resistance may not be an independent predictor of endothelial function (17–19). On the other hand, the UK Prospective Diabetes Study clearly showed that metformin intervention, aimed at improving insulin sensitivity and endothelium-dependent vasodilation, led to a significant reduction in cardiovascular events in patients with insulin resistance (20). The extent to which other interventions that improve insulin sensitivity, such as caloric restriction, physical activity, and pharmacological agents, act via NO to improve cardiovascular outcomes are issues to be addressed by future studies.

## ACKNOWLEDGMENTS

No potential conflicts of interest relevant to this article were reported.

# REFERENCES