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Keywords:

  • diabetic cardiomyopathy;
  • myocardial capillary basement membrane;
  • transgenic diabetic mice;
  • electron microscopy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ∼3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec, 296:480–487, 2013. © 2013 Wiley Periodicals, Inc.

Classical descriptions of the chronic complications of diabetes mellitus (DM) generally include diabetic nephropathy, retinopathy and neuropathy (Nathan, 1993). More recently, it has been recognized that diabetic patients are predisposed to cardiac failure and have a particularly poor prognosis following myocardial infarction. These clinical findings together with postmortem cardiomyopathic changes in the absence of coronary artery disease, led to the first description of diabetic cardiomyopathy as a clinically distinct disease (Rubler, 1972). Subsequent studies of diabetics (Bell, 1995) have identified several left ventricular abnormalities that support the existence of diabetic cardiomyopathy as a distinct primary disease (Asghar et al., 2009).

Hyperglycemia-driven mechanisms are widely regarded as causative factors in the development of diabetic complications. These include activation of protein kinase C, (Ha et al., 2001), advanced glycation end products (AGEs) (Vlassara and Palace, 2002) elevated sorbitol (Wallner et al., 2001), increased transforming growth factor β (TGFβ) activity (Ziyadeh, 2004) and oxidative stress (Giaccco and Brownlee, 2010; Kashihara et al., 2010). Most investigators believe that a combination of these processes are involved, and though it is not known which mechanism is most dominant or pervasive, it is clear that oxidative stress plays a pivotal role in the development of chronic complications of DM (Brownlee, 2005). Overall, antioxidant status is reduced in diabetes (Wolff et al., 1991; Vijayalingam et al., 1996) and ROS are important in many diabetic complications (Baynes, 1991; Low et al., 1997).

This concept is supported by the results of several of our own studies (Liang et al., 2002; Zheng et al., 2008) in which we have described the features of a transgenic diabetic mouse (OVE26, herein referred to as OVE) that provides a remarkably accurate morphological and functional model for human DM (Zheng et al., 2008, 2004; Teiken, et al., 2011, Carlson et al., in press). In his seminal work, Liang et al. (2002) showed that breeding OVE to a second transgenic mouse (Mt) with targeted overexpression of MT in the myocardium resulted in bi-transgenic (OVEMt) progeny in which the progression to advanced diabetic cardiomyopathy was markedly reduced. These findings provided evidence for a direct role for myocardial oxidative damage in diabetic mice and demonstrated that targeted MT overexpression in cardiac myocytes resulted in significant protection against several cardiopathological consequences of DM.

Because previous studies have not examined whether targeted MT overexpression may provide protection against CBM thickening, we hypothesized that CBM width would be reduced in OVEMt bi-transgenic animals. This idea was tested with a series of LM and TEM morphometric studies in which myocardial CBM width was compared in 350-day-old (1) FVB control mice, (2) Mt mice overexpressing MT in cardiomyocytes, (3) OVE transgenic diabetic mice, and (4) bi-transgenic OVEMt mice (Mt mice bred to OVE animals).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Development of Mt Transgenic Mice and Breeding to OVE Mice

OVE transgenic diabetic mice were developed in the laboratory of Dr. Paul Epstein, as previously described (Epstein et al., 1989). The cardiomyocyte-specific MT transgene, was constructed in the laboratory of Dr. James Kang, (Kang et al., 1997). Approximately 100 copies of the purified transgene were microinjected into one pronucleus of each one-cell embryo of the inbred FVB mouse strain obtained from the University of North Dakota Biomedical Research Center. Founder mice were bred with mice of the same strain, and transgenic offspring were routinely identified by PCR using a primer pair derived from the MHC promoter and human MT-IIa gene. Transgenic positive mice (heterozygotes) and negative littermates were then used for experiments. Male diabetic OVE mice were crossed with Mt mice to produce bi-transgenic OVEMt mice. All mice were produced and maintained on the inbred FVB background.

Light and Electron Microscopy

Seven FVB, four Mt, eight OVE, and seven OVEMt 350-day-old animals were sacrificed by vascular perfusion. Prior to sacrifice, all mice were weighed and percentages of HbA1c were quantified using an A1CNow+ kit (Bayer HealthCare). All mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg kg−1). The right atrium was incised to allow exsanguination and fluid drainage. Perfusates included washouts (at 4°C) of 35 mL half-strength Karnovsky's (1965) fixative with 1% procaine, and 75 mL full-strength Karnovsky's final fixative. Perfused hearts were removed and immersed in chilled Karnovsky's fixative and then immediately cut transversely to produce ventricular “rings” (1- to 2-mm wide). The rings were minced to form small (1 mm3) tissue blocks. Left ventricular blocks were further fixed at 4°C for 2–4 h, rinsed in 0.2N sodium cacodylate buffer (pH. 7.4), postfixed in 1% OsO4 (90 min), rinsed in distilled water, and stained 90 min en bloc (0.5% aqueous uranyl acetate), all at 4°C. Tissues were conventionally dehydrated and embedded in Epon/Araldite (Carlson and Hinds, 1981).

Tissue Sampling

Myocardial epoxy tissue blocks to be sectioned were randomly chosen without visual cues (i.e., all blocks were similarly shaped and equally likely to be chosen) from small containers of similar shaped samples. Accordingly, all section angles had an equal opportunity to be cut and measured and all capillary profiles were equally likely to be identified for evaluation. Toluidine blue (1% in 1% sodium borate) stained ~0.25-µm thick sections were made from the selected blocks for determination of tissue position, orientation and light micrography. All capillary profiles that were well-perfused, patent, and completely surrounded by cardiac myocytes were chosen for making thin sections for TEM observations and calculation of myocardial CBM thickness and further study. These procedures were carried out by “blinded” technical personnel who were unaware of the group being sectioned.

Thin sections (silver–gray interference color) were cut on an RMC MTXl ultramicrotome equipped with a Diatome diamond knife, collected on 300-µm mesh naked copper grids, stained with lead citrate (Venable and Coggeshall, 1965) and uranyl acetate (4% in absolute ethanol), and observed in a Hitachi 7500 TEM at initial magnifications of 4,000–15,000 diameters.

Morphometric Analysis

A modification of the harmonic mean method summarized by Dische (1992) was adopted for determining true myocardial CBM thickness. Transmission electron micrographs were made of capillaries (15,000 × initial magnification) chosen from at least six tissue blocks from each of four to eight animals of each genotype. For TEM calibration, a standard carbon cross-grating (2,160 lines mm−1) was photographed under the same conditions used for observation of tissues. All negative films were scanned and prints were made at a constant digital setting to produce identical final magnifications. Points of measurement were randomized using a clear plastic sampling grid superimposed on 20 randomly chosen micrographs obtained from epoxy blocks of each animal genotype. Myocardial CBMs were marked and their perpendicular width was measured wherever subtended endothelial cell membranes intersected sampling grid lines. Because the external limit of myocardial CBMs are not specifically delimited (e.g., by cell membranes) basal endothelial cell membranes were used as internal limits, and outer edges of the laminae densae were considered external limits.

A measuring ruler with a quasi-logarithmic scale indicating nine separate “classes” of increasing length (adapted from Hirose et al., 1982) was numerically defined in micrometers using a digitizing tablet interfaced with a computer program designed to measure length (Bioquant, R & M Biometrics, Nashville, TN). The perpendicular distance (orthogonal intercept) across BMs was recorded and sorted in ascending order by Bioquant. Numerical measurements were then converted to their corresponding classes as indicated by the measuring ruler. The harmonic mean apparent thickness [harmonic mean apparent thickness = sum of class midpoints × no. of observations\sum of observations (Dische, 1992)] was calculated (Hirose et al., 1982) and then multiplied by 8/(3π) (Jensen et al., 1979) to remove an expected right-sided skew (Gundersen and Osterby, 1972) and to yield the “true” BM thickness. The average number of measurements per animal was ~300 with a range of ~240–400.

Statistics

Distributions of true myocardial CBM thickness derived from orthogonal intercepts on sets of 20 randomized transmission electron micrographs were analyzed as previously described (Carlson et al., 2003, Greenwood et al., 2011, Teiken et al., 2011, Carlson et al., in press). Two-way analysis of variance (ANOVA) and a Holm–Sidak post hoc multiple comparison test were used to compare CBM thickness for each mouse genotype. All data are presented as mean ± the standard deviation. Significance values for all tests were set at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

General Condition of Animals

At 350 days of age, FVB and Mt mice were physically indistinguishable. They were highly active, exhibited sleek shiny coats and were approximately the same size. OVE and OVEMt mice were similar with the exception that they had substantially smaller eyes than their nondiabetic counterparts. This was a result of the coinjection and cointegration of a cataract-inducing GR19 gene inserted when the OVE mice were developed (Epstein et al., 1989). Diabetic mice weighed slightly less than FVB and Mt animals, and though most organs (liver, heart, lungs, and spleen) were approximately the same weight in all animals, OVE and OVEMt mice exhibited nephromegaly and their combined left and right kidney weights exceeded those of FVB and Mt mice by ~35–40% (Table 1). Glycated hemoglobin levels in OVE and OVEMt mice exhibited percentages ≥10.0,and were significantly higher (P < 0.05) than age-matched FVB and Mt nondiabetic controls (Table 1). Concomitant fasting blood glucose levels measured in all genotypes were consistent with the HbA1c data (data not shown).

Table 1. Body weight, kidney weight and glycated hemoglobin in 350 day-old mice
GenotypeFVBMTOVEOVEMT
  1. a

    Significantly different than OVE (P < 0.05).

Body weight (g)23.90 ± 0.5225.60 ± 1.8223.60 ± 0.9523.30 ± 1.14
Kidney weight (mg)0.36 ± 0.020.36 ± 0.020.48 ± 0.020.52 ± 0.01
HbA1c (%)4.65 ± 0.05a4.60 ± 0.10a9.84 ± 0.3010.00 ± 0.32

Light and Transmission Electron Microscopy

General myocardial morphology

Light microscopic and TEM images were made of left ventricular cardiac tissues from 350-day-old nondiabetic FVB and Mt mice and age-matched diabetics (OVE and OVEMt). At the LM level, images of longitudinal sections of left ventricular myocardium from FVB (Fig. 1A) and Mt (Fig. 1B) mice showed features considered typical for this tissue (Ross and Pawlina, 2011). These included centrally located nuclei, evenly spaced cross-striations formed by sarcomeres of regular shape and size, and densely staining cross-band attachment sites for cardiac muscle cells (intercalated discs). Similarly, at the TEM level, cardiomyocytes from control FVB (Fig. 2A) and Mt (Fig. 2B) mice showed ultrastructural features considered typical for this cell type (Ross and Pawlina, 2011) These included in-register sarcomeres delimited by in-line Z-lines, continuous parallel rows of mitochondria between cardiac myofibrils and clearly identifiable intercalated discs.

image

Figure 1. Light micrographs of representative toluidine stained thick sections from left ventricular myocardium from of FVB (A) Mt (B) OVE (C) and OVEMt (D) mouse genotypes. Longitudinal sections of left ventricular myocardium in FVB, Mt, and OVEMt mice show normal histoarchitecture with myofibrillar bundles running primarily in the same direction. Myocardial capillaries are mainly well perfused and devoid of erythrocytes. The area outlined in the OVE diabetic myocardium (C) shows random distribution of myofibrils and a lack of fibrillar parallelism. Similar dysmorphic foci are relatively uncommon but are seen more frequently in the wide areas sampled by LM than in the reduced areas imaged at higher magnifications by TEM (see Fig. 2C).

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image

Figure 2. Representative transmission electron micrographs of longitudinally sectioned myocardium from left ventricles of FVB (A) Mt (B) OVE (C) and OVEMt (D) mouse genotypes. FVB, Mt, and OVEMt (progeny of OVE and Mt cross) show normal fine structure with myofibrils comprised of regular and continuous sarcomeres. The latter are demarcated by Z lines, which are in register with adjacent myofibrils. Rows of moderately electron dense mitochondria intervene between myofibrils. OVE diabetic myocardium (C) shows a focal area of randomly distributed mitochondria between poorly organized myofibrils in an electron-lucent sarcoplasm. These are relatively uncommon in OVE mice but are not seen in myocardial thin sections of FVB, Mt, or OVEMt animals.

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By contrast, toluidine blue stained tissues from OVE mice occasionally showed focal areas of sarcomeric disorganization (~20% of thick sections) (Fig. 1C), destroying the clean side-by-side appearance of cardiac fibers seen ubiquitously in FVB and Mt samples. At greater TEM resolution, myocardial tissue from OVE animals (Fig. 2C) showed clearly the detrimental effects of diabetic cardiomyopathy. Disorganized, collections of mitochondria randomly interspersed between disrupted myofibrils in an edematous sarcoplasm were common and represented TEM images of the sarcomeric dysmorphia identified by LM (cf. Fig. 1C).

Similar areas of sarcoplasmic disorder were not identified in LM or TEM sections derived from diabetic OVEMt mice (Figs. 1D and 2D). LM and TEM images confirmed the striking protective effect of MT overexpression against the myocardial injury seen in diabetic animals (Figs. 1D and 2D). In OVEMt animals, myocardial fine structure was essentially indistinguishable from FVB and Mt controls. Myofibrillar injury was not identified in these tissues.

Myocardial capillary basement membranes

Uninterrupted densities representing myocardial CBMs were not distinguishable in toludine blue sections (Fig.1) or low magnification TEMs (Fig. 2). However, at initial magnifications of 15,000 diameters, myocardial CBM laminae densae were clearly identifiable by TEM (Figs. 3A–D). Cursory visual observation showed only small differences in CBM thickness of the four animal genotypes. Accordingly, to measure the CBM widths, we chose the unbiased orthogonal intercept method (Jensen et al., 1979), which is considered the gold standard for measuring BM width. As expected, our data showed that left ventricular myocardial CBMs in OVE diabetic mice were increased in width. At 350 days of age, mean myocardial CBM thickness in OVE diabetic animals (n = 8) was 62.18 ± 10.34 nm. This was significantly thicker (~28%, P < 0.05) than comparable CBMs in nondiabetic FVB controls (n = 7), which averaged 48.50 ± 7.31 nm. Moreover, OVE myocardial CBMs were significantly wider (~40%, P < 0.05) than comparable CBMs (44.56 ± 3.62 nm) in Mt animals (n = 4). However, by far the most remarkable finding was the mean myocardial CBM thickness in diabetic OVEMt (n = 7) mice (47.28 ± 7.07 nm). This was >31% thinner (P < 0.05) than its OVE counterpart, and >3% thinner than age-matched FVB controls.

image

Figure 3. Representative transmission electron micrographs of left ventricular myocardial capillary endothelial lining and subjacent CBMs of 350-day-old FVB (A), Mt (B), OVE (C), and OVEMt (D) mouse genotypes. Opposing arrows in insets (upper right) indicate measured width of CBMs at 2.6× diameters of landscape micrograph.

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Two-way ANOVA and post hoc Holm–Sidak tests showed that as expected, diabetes, and diabetes alone (OVE) was sufficient to increase the basement membrane thickness significantly relative to all other genotypes. Moreover, in the OVEMt genotype, Mt alone was sufficient to significantly protect against diabetes-induced increased basement membrane thickening.

Because CBM thickening is nearly ubiquitous in diabetic patients and animals, and is considered the virtual hallmark of diabetic microvascular disease, its absence in OVEMt animals was unexpected and striking. To our knowledge, morphometrically confirmed elimination of increased BM width in diabetics has not been reported previously, and relative to OVE diabetic mice, elimination of diabetes-induced increased CBM thickness was a new finding (Fig. 4).

image

Figure 4. Morphometric differences in myocardial CBM thickness ±S.D. at 350 days of age: FVB, Mt, and OVEMt versus OVE all show statistically significant differences (P < 0.05). OVE mice show the thickest myocardial CBM, while OVEMt mice show substantial cardioprotection against myocardial CBM thickening and of the four genotypes studied, OVEMt mice exhibit the narrowest myocardial CBMs.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The objective of the current study was to quantify myocardial CBM thickness in four age-matched mouse genotypes. We found that targeted overexpression of MT in cardiac myocytes eliminates the structural damage in these cells, a finding consistent with those demonstrated by Liang et al. (2002).

Capillary BM thickening was not measured until 1968, when Siperstein et al. demonstrated in skeletal muscle that CBM thickening was characteristic of nearly all patients (98%) with blood glucose values >140 mg dL−1 (Siperstein et al., 1968). Subsequent development of TEM techniques provided the resolution necessary to demonstrate changes in the diabetic microvasculature including thickened CBMs. It is currently widely accepted that accelerated CBM thickening is a diagnostic feature of advanced stages of DM (Viberti, 1994).

As part of an ongoing comprehensive morphometric analysis of the 350-day-old mouse heart in four different genotypes, the current study centered specifically on left ventricular myocardial CBMs. The data showed that CBM thickness in OVE diabetic animals averaged 62.18 ± 10.34 nm, while the mean of FVB controls was 48.5 ± 7.31 nm. This relatively large (28%) difference in diabetics versus controls confirmed that myocardial CBM is one of the most susceptible to hyperglycemia-driven thickening (Carlson et al., 2003). Much more remarkable, however, was our finding that CBM widths were essentially identical in FVB and OVEMt animals, indicating that myocardial CBM thickening in animals with cardiomyocyte targeted MT overexpression was not simply reduced, but rather was completely abolished.

A number of molecular mechanisms have been implicated in the pathogenesis of diabetic BM disease and a unifying hypothesis suggests that since hyperglycemia increases free radical generation, ROS may be significant mediators of diabetic tissue injury, and their increased production may represent a final common pathway toward chronic complications of the disease (Brownlee, 2005; Giaccco and Brownlee, 2010). In this regard, data in the current study strongly implicate a direct role for oxidative damage in the initiation of a pathway to myocardial injury and associated CBM thickening.

Although the molecular mechanisms responsible for diabetic CBM thickening are poorly understood and likely are multifactorial (Tsilibary, 2003), most theories invoke a form of decreased ECM turnover resulting in increased BM width. In this regard, the advanced glycation end product (AGE) hypothesis (Brownlee et al., 1984) is attractive. This concept centers on non-enzymaticglycation of proteins and other long-lived molecules, the formation of which is regulated by glucose concentration and exposure time (Vlassara and Palace, 2002) and could lead to reduced CBM turnover resulting from decreased degradation of highly cross-linked long-lived proteins such as collagen and other ECM proteins (Brownlee et al., 1984). However, formation of AGE also is believed to play a central role in increased oxidative stress and upregulated ROS (Goh and Cooper, 2008) and it is well-known that increased ROS activate various transcription factors and cytokines leading ultimately to increased expression of ECM genes, fibrosis, and thickened BMs (Mason and Wahab, 2003).

Although additional confirmation is required, current data strongly support the concept that intracellular ROS have a direct role in overproduction of ECM proteins (Ha and Lee, 2000; Brownlee, 2001). Furthermore, it has been shown that ROS activate a cascade of downstream reactions in which TGF-β and connective tissue growth factor coordinate to promote upregulation of transcription of numerous matrix genes (Nath et al., 1998; Iglesisus–de et al., 2001; Park et al., 2001), and repress that of matrix metalloproteinases—concomitant events which in vivo could lead symbiotically to decreased CBM turnover (Mason and Wahab, 2003). It is possible, therefore, that increased CBM width in diabetic animals could result either from hyperglycemia-driven ROS production and increased ECM synthesis, or decreased degradation driven by inhibited matrix metalloproteinases and inexorably increasing highly crosslinked AGE products. Alternatively, both mechanisms could be operable.

Chronic complications of diabetes exhibit a series of common morphological features including interstitial fibrosis, arteriolar and microvascular BM thickening. These structural changes occur relatively late in the course of the disease, and it seems likely that they may represent the consequences of hyperglycemia-driven oxidative stress. In a recent review, Kashihara et al. (2010) point out that in the progression to diabetic complications, numerous opportunistic macromolecules, including NAD(P)H oxidase, AGE, polyol pathway defects, uncoupled nitric oxide synthase (NOS) and oxidative phosphorylation have been implicated in the increased production of ROS and subsequent tissue damage. This interpretation is consistent with those of a number of investigators who postulate that oxidative stress is a common denominator for the pathways involved in the progression of chronic complications of diabetes (Prabhakar et al., 2007; Giacco and Brownlee, 2010; Kashihara, 2010). It follows therefore, that mechanisms leading to reduced ROS, could be expected to be protective against redundant production of ECM proteins, including those comprising myocardial CBMs.

Although MT is a group of intracellular metal-binding and cysteine-rich proteins that function primarily as metal homeostasis regulators, in the 1980s they were found to be potent antioxidants (Onosaka et al., 1988). Subsequent in vitro studies showed they reacted directly with ROS. Significantly, multidisciplinary investigations have shown that MT also function to protect against oxidative injury in vivo. It is not surprising, therefore, that the antioxidant function of MT has been explored extensively (Li et al., 2007) and in several important studies in which MT was overexpressed in the diabetic myocardium (Cai and Kang, 2001; Liang et al., 2002; Ye et al., 2003) ROS was apparently mitigated and chronic complications were effectively reduced. More recently it was shown that the MT prevention of diabetic cardiomyopathy is mainly due to its suppression of diabetes-derived nitrosative stress and damage (Cai, 2006).

Cardiac myocyte structure and function were the focus of most previous investigations of MT overexpression in the myocardium (see Li et al., 2007 for review), and it was assumed that the demonstrated MT protective function correlated closely with inhibition of ROS-induced lipid peroxidation. However, the current study centers on myocardial capillaries in similar animal genotypes and uncovers a previously unrecognized literal elimination of hyperglycemia-driven CBM thickening, the mechanism of which is not clear but appears to be directly related to ROS mitigation by MT.

In summary, the current myocardial study strongly supports an hypothesis that in response to hyperglycemia-driven oxidative stress, CBM-associated cells trigger a series of early molecular microangiopathic events leading progressively to increased ROS and subsequent sequential diabetic complications including significantly thickened myocardial CBMs. The data also indicate that MT overexpression in cardiac myocytes, and possibly other cell types is fully capable of abolishing an otherwise expected significant diabetic CBM thickening.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The authors gratefully acknowledge the superb electron microscopic technical assistance of Laurie Kim Young. They thank Dr. Patrick Carr for expert assistance with statistical analysis. There are no potential conflicts of interest regarding financial and personal relationships between any of the authors and other individuals that might bias their work. Also, no part of this manuscript has been submitted or is currently being considered for publication elsewhere. The ethical background for this study derives from an ethically neutral hypothesis and all data collection and interpretation are believed to have been carried out in the absence of unethical behavior. No ethical committee has reviewed the manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED
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