Vascular complications are associated with the progressive severity of diabetes, resulting in significant morbidity and mortality. This study quantifies functional vascular parameters and macrovascular structure in a rat model of type 1 diabetes. While there was no difference in the systemic arterial elastance (Ea) with 50 days of diabetes, changes were noted in the aorta and femoral artery including increased tunica media extracellular matrix content, decreased width of both the media and individual smooth muscle cell layers, and increased incidence of damaged mitochondria. Extracellular matrix proteins and elastin levels were significantly greater in the aorta of diabetic animals. These differences correlated with diminished matrix metalloprotease activity in the aorta of the diabetic animals. In conclusion, diabetes significantly altered the structure and ultrastructure of the aorta and femoral artery before systemic changes in arterial elastance could be detected.
The worldwide incidence of type 1 diabetes is increasing at a rate of 3% per year [
Clinical tools used to assess large vessel pathology indicate that vessel wall structure and stiffness change early in the progression of diabetes [
A change in arterial mechanical properties can occur with increased collagen cross-linking as a result of glycation [
The purpose of this study was to determine the morphometric characteristics of diabetes-induced abnormalities of two major vessels, the aorta and the femoral artery, at an early time point before functional changes were noted at the systemic level and follow those changes as the disease progressed.
Thirty-six male Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 250–270 grams were divided into two groups: diabetic and nondiabetic control (
Development of diabetes was determined by observing blood glucose levels greater than 300 mg/dL as measured by an Accu-Check Advantage glucometer (Boehringer Mannheim Corporation, Indianapolis, IN). Rat body weights and blood glucose levels were recorded weekly. Rats were provided free access to food and water, and principles of institutional laboratory animal care were strictly followed. The rats were sacrificed with an intraperitoneal injection of pentobarbital.
Functional evaluation was performed using left ventricular catheterization through the right carotid artery with a 2 French Millar microtip pressure volume catheter (Millar Instruments, Houston, TX) as previously described [
Following animal sacrifice, one-to-two millimeter segments of thoracic aorta and proximal sections of the femoral artery were immediately resected and rinsed in ice-cold phosphate-buffered saline. Samples were gathered from the center sections of the vessels and quickly immersed in fixative at +4°C.
Samples of thoracic aorta and femoral artery were fixed in 4% paraformaldehyde and stored at +4°C. Tissues were processed by rinsing with water before undergoing dehydration through a graded ethanol series ending in xylene. Samples were embedded in paraffin. Five micrometer paraffin sections were cut and stained with hematoxylin and eosin (H&E) or Masson’s trichrome. Vessels from three rats in each group were used for calculations. Digital images were acquired using a Nikon Eclipse TE300 microscope attached to a SPOT 32 system (Diagnostic Instruments, Inc., Sterling Heights, MI).
All analyses were blinded and completed on digital images using Adobe Photoshop (Adobe Systems Inc.) or Scion Image (Scion Corporation). Vessel width was measured at 10 evenly spaced points along the vessel at 20x. All other analyses were completed on images taken at 40x magnification. The SMC layer width was measured at 10 evenly spaced sites between elastic bands along the vessel length of all but the first and last layers within the media to eliminate any sectioning artifacts or subsequent misidentification of endothelium or adventitia. Total vessel area measurement including intima, media, and adventitia and arterial ECM content was calculated by color analysis from sections stained with Masson’s trichrome.
Tissues were fixed in 2% glutaraldehyde, rinsed in PBS and postfixed with 1% osmium tetroxide. Rinsing with distilled water was followed with a graded ethanol dehydration series ending with propylene oxide. The samples were infiltrated using a mixture of 1/2 propylene oxide and 1/2 resin overnight and then embedded in Epon 812 resin (Electron Microscopy Sciences, Ft. Washington, PA). One-micron-thick sections of the vessel were examined under the light microscope before 80 nm sections were cut on an LKB Nova Ultratome. Thin sections were captured on precleaned copper grids and stained using a double lead stain technique [
Lipid incidence, mitochondrial area, and mitochondrial membrane integrity were assessed from electron microscopy (EM) photomicrographs taken at final magnifications of 8,700x and 21,600x. To assess mitochondrial quality, each mitochondrion was graded as intact or compromised using previously published techniques [
Aorta was removed from a STZ-injected rat at 50 days of diabetes and ~8 mm piece was cut and rinsed in ice cold 0.15 M NaCl. The tissue was homogenized using Polytron homogenizer in an ice-cold extraction buffer (1 : 3 wt/vol) containing the following: 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35, and 0.02% NaN3. The homogenate were shaken on cold overnight, then centrifuged at 4°C for 30 min at 12,000 ×g and the supernatants were collected. Protein concentration was measured using Protein Assay Reagent (Bio-Rad, Hercules, CA).
In [
Quantitative analysis of the ultrastructure was completed on photomicrographs taken from a minimum of five independent regions within a section from each sample at each magnification aforementioned. Statistical analysis using Sigma Stat software (SPSS, Inc.) employed
The body weight of the STZ-diabetic rats and matched controls was not different at the initiation of the study. The control animals weighed
Nonfasting blood glucose levels, while between 101–113 mg/dL in control rats, increased 471% with 50 days of diabetes, 509% with 100 days of diabetes, and 407% with 150 days of diabetes. The high glucose levels in the diabetic rats were consistent across the weekly measurements. The average blood glucose levels at the time of termination are presented in Table
Mean rat blood glucose levels: mean nonfasting blood glucose levels show elevated readings for the diabetic groups throughout the duration of the study (
Blood glucose | Control | Diabetic |
---|---|---|
50 day | ||
100 day | ||
150 day |
*indicates
There was no statistical difference in resting blood pressure recordings, obtained under anesthesia, for the diabetic group compared to the matched controls. With 50 days of diabetes the mean blood pressure of the control group was
Arterial elastance was measured via catheters placed within the left ventricle. No difference was detected between the control and diabetic groups (
H&E-stained sections illustrated differences between diabetic and control vessels in the intima-media width, and the width and number of SMC layers (Figures
Diabetes-induced changes in the aorta: vessel (intima-media) width was measured as shown in Figure
Duration of diabetes | ||||||
Experimental group | 50 days | 100 days | 150 days | |||
Control | Diabetic | Control | Diabetic | Control | Diabetic | |
Vessel width (mm) | ||||||
SMC area (percent of vessel wall, including adventitia) | ||||||
Heterochromatin (% of nuclear area) | ||||||
Invaginations per nuclear envelope | ||||||
SMC area/ECM area (ratio) | ||||||
ECM (percent of vessel wall area) |
Diabetes-induced changes in the femoral artery: mimicked the changes in the aorta with the exception of the SMC/ECM area which was statistically higher in the femoral artery of the 50-day diabetic animals, but not in the aorta. Heterochromatin was less in the diabetic samples at 150-days. Lipid droplet density was statistically greater in the diabetic group at 150 days. Finally the elastin band width was greater in the diabetic group throughout the study duration.
Duration of diabetes | ||||||
Experimental group | 50 day | 100 day | 150 day | |||
Control | Diabetic | Control | Diabetic | Control | Diabetic | |
SMC area (percent of vessel wall) | ||||||
SMC area/ECM area (ratio) | ||||||
Nuclear/cytoplasmic area (ratio) | ||||||
Heterochromatin (% of nuclear area) | ||||||
Invaginations per nuclear envelope | ||||||
Lipid Droplets/area | ||||||
Elastin band width |
Morphological duration-dependent changes in the aorta of diabetic rats. Control aorta showed a typical H&E staining pattern with SMCs and elastic layers staining pink (lighter and darker, resp.) while nuclei were purple. At the 150-day time point, the SMC layers became slightly less defined and the vessel wall was reduced in cross-sectional area. The measured vessel wall width (tunica intima-media width) is illustrated by the gray arrow in the 100-day control image. An example of the SMC layer width measurements is shown with a solid black line in the 100 day control image (Magnification: 40x).
Tunica media characteristics in aorta. (a) The media width was significantly reduced in the diabetic aortas when compared to the control aortas at all three time points (
The number of SMCs per layer, indicative of cellular proliferation, was significantly higher at 50 and 100 days in the diabetic rats when compared to their respective control groups (
In control animals, aortic SMCs formed a distinct and nicely organized arrangement (Figure
Examples of electron micrographs of aorta. (a) EM micrographs (8,700x magnification) showed a distinct organization of SMCs within each layer, sandwiched by elastic bands (El). The arrowed line in the 50 day control image illustrates a typical elastic band width measurement. In the diabetic aorta, patches of SMC disorganization were evident. Nuclei (N) of cells from diabetic animals often appeared to have a different spatial orientation with invaginated membranes (50 days). The cytoplasm (Cy) was more spread out with finger-like projections in the diabetic samples. The matrix surrounding the SMC contained a mixture of collagen (Col) and elastin (El). (b) Micrographs (21,600x magnification) show SMC nuclei (N) surrounded by cytoplasm (Cy) either rich in cytoskeletal fibers (clear
An increase in the ratio of nuclear to cytoplasmic area has been used as a basic indicator of cellular hypertrophy [
Nuclear/cytoplasmic cross-sectional area. Evaluation of the electron micrographs demonstrated that the ratio of nuclear/cytoplasm area remained constant at the 50- and 100-day time points. At 150 days, the ratio significantly increased in the SMC from the diabetic rats (
While the shape of the nucleus appeared to change with extended diabetes (Figure
With 150 days of diabetes, significant portions of mitochondria were swollen or disrupted giving a vacuolated appearance to the SMC cytoplasm (Figure
Mitochondrial membrane disruption in aorta. Mitochondria were graded on the basis of membrane intactness with grade 1 as a completely intact organelle. Grade 2 indicated disruption of the outer mitochondrial membrane. Grade 3 indicated disruption of the inner mitochondrial membrane, and grade 4 meant that both membranes were disrupted. In the SMCs from control animals, the mitochondria were predominantly intact throughout the course of the study. In contrast, in the SMCs from the diabetic animals there were more grade 2–4 mitochondria present at day 50 and the percentage of disrupted mitochondria grew over the 150 day diabetes duration until over half of the mitochondria determined to be grade 3-4. Grade 3 measurements were more rare and shown as a thin sliver in the diabetic 150-day figure (
Although there were trends toward more lipid droplets in the aortas of the 50 and 100 day diabetic rats compared to the respective controls, the difference was not statistically significant until 150 days (
Lipid droplets in aorta. (a) Arrows indicate lipid droplets in a diabetic aortic sample. (b) The number of lipid droplets within the aortic SMC increased in the 150 day diabetic and control animals (
Perhaps the most dramatic changes in the diabetic vessels were noted in the ECM. Masson’s trichrome-stained sections demonstrated differences in ECM amount found within the SMC layers and in the adventitia (Figure
ECM in aorta. (a) Masson’s trichrome-stained imaged demonstrated that the amount of ECM (blue staining) increased with time in both control and diabetic aortas (magnification = 40x). (b) The increase in ECM fractional area was significant for the diabetic group at 150 days (
Changes in the area of elastic components were assessed by measuring the elastic component width (Figure
Extracellular matrix. (a) The elastic band width, measured from EM sections, was significantly higher in all diabetic samples (
In order to determine whether the diabetes-induced increase in ECM levels noted in Figure
In this study, structural changes were identified in the large vessels of a rat model of type 1 diabetes. The structural changes were present before any changes in resting blood pressure or arterial elastance were detected. Some of the most dramatic changes were found in the width of the media tunica layer of the vessel walls, in the percentage of intact mitochondria, deposition of intracellular lipid droplets, and in the extracellular matrix.
Without diabetes, the structure of the vessels changed very little over the 100 days monitored. The SMC layer width increased over time, while the elastic layer width decreased, thus keeping the media width constant. The quality of mitochondria in the SMCs from control animals progressively worsened over time, suggesting that the changes noted in the control and diabetic animals were not an artifact of tissue fixation, but associated with the aging and the disease process. Most of the changes observed in ultrastructure as a function of time were in the extracellular matrix, particularly collagen. While the elastin steadily declined, the collagen content increased over time; an alteration known to increase vessel stiffness. In humans the age-related decline in the mechanical properties of the aorta and resistance arteries includes central aortic compliance and pulse wave velocities [
Vascular changes associated with diabetes have been described as accelerated aging [
Measurements of stiffness and vascular compliance are important measures for people with type 1 diabetes. Recently, structural changes in the carotid arteries of type 1 diabetics without overt vascular disease were examined according to age and the duration of diabetes [
Aortic stiffness in people with type 1 diabetes without hypertension correlated with a decline in brain white matter, as measured by MRI. In fact aortic stiffness predicted white matter atrophy [
Most of the diabetic characteristics increased with the duration of diabetes in our model. However, a few reversed that trend including the number of SMC SMC/media layer, which showed a statistical difference at the 50- and 100-day mark, but not at 150 days. Caution should be used when evaluating these changes as only 3 animals per group survived 150 days with uncontrolled diabetes. While the effect size was large for most of the diabetic-induced changes in vessel characteristics, a lack of statistical difference in a small sample size may not be predictive of a larger population. One interesting correlation was the drop in blood glucose levels in the diabetic rats at 150 days. It is possible that certain morphological features are exquisitely sensitive to the blood glucose level, thus explaining why some parameters were not statistically different at the late time point. An additional explanation for the plateau effect may be in the general physiological condition of the surviving diabetic rats. Twenty-one weeks (150 days) is an extensive period of time for an animal to survive with uncontrolled diabetes. Finally, it is possible that there is truly a cellular plateau or cellular adaptation at which no further morphological changes can occur.
This study assists in defining the diabetic time-dependent alterations in macrovascular structure and cellular function. At the structural level we determined that with the progression of diabetes, ECM content becomes higher in the media while smooth muscle cell layers and number of smooth muscle cells per layer decline. Ultrastructural changes show increased cellular hypertrophy and alterations in mitochondria morphology with diabetes. There was an overall age-dependent increase in media lipid droplets, but with a much higher incidence in the diabetic animals and an age-dependent change in SMC nuclear morphology not related to diabetes. How any one of these observations contributes to the development of diabetic macrovascular disease will keep the field of diabetes vascular research busy for a very long time.