Background
While improved care for people with diabetes has reduced the overall risk of complications and mortality, rates of mortality for people with diabetes is still two fold higher than those without diabetes [
1,
2]. Cardiovascular disease accounts for a significant percentage of deaths of individuals inflicted with type 1 diabetes [
3‐
7]. Even in young adults, cardiovascular events such as stroke are higher in people with type 1 diabetes than in those without diabetes [
8]. As the duration of life with diabetes increases, so increases the risk of cardiovascular events and mortality [
9]. Current management procedures focusing on tight glycemic control may have only marginal effects to halt the progression of large vessel disease [
10].
While the majority of research effort concerning diabetic vascular disease has been focused on changes in the endothelial cells, alterations in the vascular smooth muscle cells (VSMCs) are also known to occur [
11‐
13]. The VSMCs surrounding vessel walls, undergo increased proliferation, adhesion, and migration in conditions of hyperglycemia [
14‐
17]. Several proteins and genes have been proposed as targets for hyperglycemic changes in the VSMCs including protein kinase C (PKC). Interestingly, many of them have a common upstream pathway that includes the release of calcium (Ca
2+) from intracellular stores, including the sarcoplasmic reticulum (SR).
In VSMCs there are two channels that have been identified as important in the release of intracellular Ca
2+ stores; the inositol 1,4,5-trisphosphate (IP
3)-sensitive store and the ryanodine-sensitive store [
18]. In VSMCs, IP
3 release of Ca
2+ from intracellular stores is a common pathway for activation of many proteins including PKC [
19] and PLC. Changes in basal levels of intracellular Ca
2+ with diabetes are inconsistent, but Ca
2+ transients in response to agonists such as angiotensin II are clearly attenuated in VSMCs from diabetic animals [
20]. The blunted IP
3-induced Ca
2+ responses could be due to changes in the intracellular Ca
2+ channels, the intracellular Ca
2+ storage site (SR), or the sarcoplasmic/endoplasmic Ca
2+ ATPase (SERCA). This study examines changes in the intracellular Ca
2+ regulatory proteins in two animal models of type 1 diabetes. The streptozotocin-induced diabetic rat is a well-established rat model using a toxin to destroy the pancreatic beta cells. The second model utilizes the DR-BB rat, which better mimics the autoimmune component of type 1 diabetes. Results from these animal models were compared to a cell culture model (A7r5) with hyperglycemia.
Materials and methods
Induction of Diabetes
Streptozotocin Rat Model
Seven week old male Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 250-270 grams were injected intraperitoneally with 65 mg streptozotocin (STZ, Sigma, St. Louis, MO) per kg body weight (n = 15). Non-diabetic control rats (n = 12) were injected with vehicle (citrate buffer). Blood glucose levels greater than 250 mg/dL, measured by an Accu-Check Advantage glucometer (Boehringer Mannheim Corporation, Indianapolis, IN), indicated the development of diabetes.
The blood glucose levels and body weights of all rats were assessed weekly. The final weight and blood glucose levels are shown in Table
1. Free access to food and water was supplied. The principles of institutional laboratory animal care were strictly followed. The rats were killed with an intraperitoneal injection of pentobarbital after 8 weeks of diabetes.
Table 1
Characteristics of Animal Models of Diabetes
Weight (g) | 453 ± 16 | 328 ± 14 * | 217 ± 5 | 212 ± 19 |
Blood Glucose (mg/dL) | 113 ± 5 | 554 ± 13 * | 98 ± 2 | 295 ± 13 * |
DR-BB Rat Model
Diabetes was induced in 21-26 day old Diabetes Resistant Bio-Breeding (DR-BB) Wistar rats using anti-RT6 monoclonal antibody (Dr. Dale Greiner, University of Mass) DS4.23 hybridoma in 2 ml of tissue culture medium injected 5 times/week in combination with polyinosinic-polycytidylic acid (Sigma), a non-specific immune system activator at 5 μl/g of body weight 3 times/week. Induction treatment was discontinued at the onset of diabetes as determined by a blood glucose level above 250 mg/dL. Diabetes typically occurred 2-3 weeks after the treatment was initiated. Within 1 week of diabetes development, the diabetic rats were treated with insulin (Humulin R, Humulin U and Humulin N; Eli Lilly, Indianapolis, IN) through subcutaneous implantation of an insulin-filled Alzet osmotic pump (Alza, model 2004, 0.25 μl/hr for 28 days; Mountain View, CA). Blood glucose was closely monitored for the remaining time, and hyperglycemic animals were given insulin injections as needed in amounts based upon the severity of hyperglycemia. Control rats underwent the same manipulations except pumps were filled with saline. Body weight and blood glucose were followed weekly, with the final values shown in Table
1. Diabetic animals (n = 7 female, 4 male) and their age matched controls (n = 6 female, 4 male) were euthanized after 8 weeks of diabetes.
Cell Dispersion
Dissected aortas and femoral arteries were cleaned of adventitia in cold phosphate buffered saline (PBS) buffer. The vessels were cut lengthwise to expose the intima, which was gently swabbed to remove endothelial cells. Each vessel was pinned, lumen side up, in a small covered beaker containing Sylard Gel (Dow Corning, Midland, MI) and bathed in collagenase solution containing deoxyribonuclease I, collagenase type 2, soybean trypsin inhibitor (Worthington Biochemical Corporation, Lakewood, NJ) and bovine albumin (Sigma). After 30 minutes of shaking at 37°C, the first digest was discarded as it contained predominantly endothelial cells. New collagenase solution was added and further dispersions were gathered every 2 hours. Cells were gently centrifuged to remove the enzyme solution before being rinsed and re-suspended in media.
Cell Culture
Cells from the A7r5 cell line (American Type Tissue Culture, Manassas, VA), derived from rat thoracic aorta, were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C, 5% CO2. Cells were grown in flasks, then seeded in 100 mm dishes or on coverslips with glucose concentrations of either the supplier-recommended concentration (25 mM; referred to as medium) or high glucose (75 mM). Because 25 mM glucose is high compared to physiological serum levels, a third (low) glucose concentration was tested (5.5 mM). The effects of osmolarity were controlled by the addition of mannitol. Cells were grown to semi-confluence prior to testing.
Immunoblotting
Total proteins were extracted from tissue or cell culture using lysis buffer containing 10 mM Tris, pH 7.4, 1 mM sodium ortho-vanadate and 1% SDS. To separate nuclear and cytoplasmic proteins, the NE-PER kit (Pierce, Rockford, IL) was used. Each sample was concentrated by ultrafiltration using Microcon YM-10 (Millipore, Bedford, MA). Protein concentrations were measured in 96-well format using the BCA kit (Pierce, Rockford, IL) and a MRX microplate reader (Dynex Technologies, Chantilly, VA). A total of 30 or 50 μg of protein was loaded in each lane of 8, 10 or 4-15% gels. Rat cardiac tissue extract was used as a positive control for ryanodine receptor (RyR) and A-10 cell lysate (Santa Cruz, Santa Cruz, CA) was used for Inositol 1,4,5 trisphosphate receptors (IP3Rs). After electrophoretic separation, proteins were transferred from a gel to a PVDF membrane (Pierce) overnight. Ponceau's stain (Sigma) was used to assess protein loading prior to blotting. A standard immunoblotting protocol was performed. Primary antibodies to SERCA2, SERCA3, IP3R type I and RyR (Affinity BioReagents, Golden, CO), and IP3R type 2 and IP3R type 3 (Santa Cruz) were used. GAPDH was used as a housekeeping gene product (antibody, Santa Cruz). Anti-HSP90 (Santa Cruz) was used to assess the purity of the cytoplasmic extractions and anti-p62 (BD Transduction, Lexington, KY) or anti-USF-2 (Santa Cruz) were used to test the purity of the nuclear extracts. Detection was performed using enhanced chemiluminescent reagent (Pierce) or SuperSignal West Pico Chemiluminescent reagent (Pierce). Duplicates were made of each immunoblot experiment.
Immunocytochemistry
A7r5 cells were grown on cover slips in various glucose concentrations. Freshly dispersed VSMC were allowed to attach to glass coverslips for at least 2-4 hours in PBS at 37°C prior to fixation with 2% paraformaldehyde (Fisher Scientific, Palatine, IL). Fixed cells were rinsed with PBS, permeabilized with 1% Triton X-100 (Sigma) and rinsed well before blocking in 10% goat or donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA). Cells were incubated with primary antibody (same as above) diluted in 5% non-fat dry milk (Bio-Rad, Helcules, CA) solution overnight in a cold box at manufacturer recommended concentrations. Alpha smooth muscle actin (Sigma) was used to identify SMC type. Cells were repeatedly rinsed in PBS before incubation with secondary antibody conjugated to Rhodamine Red or Cy 2 (Jackson ImmunoResearch) in a dark chamber. Brefeldin A (Invitrogen, Carlsbad, CA) was used to evaluate sarcoplasmic reticulum (SR). Coverslips were rinsed and allowed to air dry in the dark before mounting. Images were captured using an Olympus Fluoview 300 Confocal Microscope with the consistent confocal settings for each pair of control and diabetic cells per single primary antibody. Experiments were performed in triplicate.
Live Cell Intracellular Calcium Measurements
Freshly dispersed VSMCs or cultured A7r5 cells were allowed to attach to coverslips for 1 hour in PBS at 37°C before loading with 1 μM each of two Ca2+ sensitive fluorophores, Fluo-4/AM and Fura-Red/AM (invitrogen) by incubation at 37°C for 30-60 minutes. The coverslips were inserted into an Attofluor chamber (Molecular Probes) and rinsed with warm PBS. PBS with ethylene glycol tetraacetic acid (EGTA) to buffer extracellular Ca2+ (0 Ca2+), was added to the chamber mounted on the stage of a confocal microscope so that the measured Ca2+ changes were limited to the intracellular stores only. Baseline measurements were taken on quiescent cells for 3-4 minutes prior to the application of any agonist. Vasopressin (10 nM, Sigma) or thapsigargin (20 μM, Sigma) were added to the bath at the times indicated in the results section. Images were collected every 5 or 10 seconds using an inverted Nikon Eclipse TE 300/Bio-Rad MicroRadiance Plus Laser Scanning Confocal Microscope (Bio-Rad Laboratories, Germany) or an Olympus Fluoview 300 confocal microscope (Olympus, Center Valley, PA). Fluoview software was used to analyze Ca2+ signaling experiments. Time-to-peak was defined as the time of agonist application to the time the maximal magnitude of the response was obtained. The duration of the Ca2+ signal was defined as the time from the upstroke of the response to the return within 25% of initial baseline value.
Data analysis
All data points were normalized for background signal prior to statistical analysis. Fluoview software (Olympus, Melville, NY) was used to analyze Ca2+ signaling experiments. Adobe Photoshop (Adobe Systems, San Jose, CA) was used to analyze relative fluorescence intensity, and color analysis. Analysis of variance was performed for Ca2+ fluorescence and immunoblot density values using Sigma Stat software. When appropriate, statistical differences were assessed using Dunnett's test for multiple comparisons after a one-way analysis of variance using Sigma Stat (SPSS Inc, Chicago, IL). A probability level of p < 0.05 was defined as a significant difference.
Discussion
VSMC dysfunction and subsequent disease is the main cause of mortality in type 1 diabetes, but the underlying mechanisms are largely unknown. In normal (medium) glucose conditions reported here, the distribution of the IP
3R subtypes, RyR, and SERCA were similar to other studies using primary aortic SMCs [
23], rabbit aortic SMCs [
24], and cultured cell lines [
25]. Studies involving rat heart [
26], islet cells and kidney [
27] using diabetic animal models have demonstrated alterations in IP
3R-1, SERCA2 and SERCA3 expression in a manner very consistent with our results from VSMCs.
The results of this study clearly demonstrate changes in the regulatory proteins associated with intracellular Ca2+ storage and release in aortic and femoral VSMCs. These changes lead to altered Ca2+ signaling in the cytoplasmic and nuclear compartments in response vasopressin. VSMCs from two different diabetic rat models showed a decline in the levels of IP3R and SERCA protein with diabetes. Changes in the distribution of the IP3R subtypes and SERCA subtypes were dependent on the rat model, but fully supported the immunoblot results of overall decreased amounts. To measure the relative role of hyperglycemia on the above mentioned changes, rat aortic cultured cells (A7r5) were exposed to various concentrations of glucose. Like the freshly dispersed cells from the diabetic animals, cultured cells exposed to high glucose had blunted Ca2+ responses to vasopressin and decreased levels of Ca2+ regulatory proteins. Thus, the changes noted in vSMCs from diabetic animals were likely due to exposure of the vSMCs to hyperglycemic conditions rather than adaptation of the cells to the complex disease of diabetes. This is the first study to report of both diminished levels and subcellular redistribution of key Ca2+ regulatory proteins in cultured and freshly dispersed VSMCs.
Previous studies have described a blunted Ca
2+ response in VSMCs from diabetic animals [
28‐
30]. Unique to this study was the identification of separate nuclear and cytoplasmic responses, and the presence of spontaneous nuclear Ca
2+ oscillations. Such oscillations in A7r5 cells have been reported to be associated with intracellular Ca
2+ stores and entry of Ca
2+ from the extracellular milieu [
31,
22]. Others have shown spontaneous Ca
2+ sparks occurring around the nucleus of primary VSMCs [
32]. The effect of glucose on the spontaneous nuclear oscillations has not been previously published. In this study, the high magnitude spontaneous oscillations in resting cells bathed in high glucose were intracellular Ca
2+ release events as all experiments were performed in zero extracellular Ca
2+.
In order to thoroughly examine the effects of hyperglycemia on VSMC function, two different animal models of type 1 diabetes were utilized; the toxin-induced STZ diabetic rat and the immune/inflammation model of type 1 diabetes, the DR-BB rat. The differences noted between the two models were minor, but in general the diabetes induced changes were more robust in the DR-BB rats. It is possible that any small differences we noted between rat models were a function of the rat strain, the method of diabetic induction, or the administration of insulin to the DR-BB animals.
In VSMCs, much of the ability to remove Ca
2+ from the cytoplasm is dependent on the SERCA proteins on the SR and nuclear envelope, estimated to be responsible for over 40% of the Ca
2+ buffering in VSMCs [
33,
34]. Importantly, by maintaining low intracellular Ca
2+, SERCA inhibits migration of VSMCs into the intima of the vessel [
35], a common hallmark of diabetes-induced vascular disease. In general, SERCA2 and SERCA3 protein levels were decreased and their distribution altered in the diabetic rats. Brefeldin A staining of the SR did not show a corresponding loss or reorganization of the SR with hyperglycemia, suggesting that these were pump-specific changes. In contrast to a previous report [
36], we did not measure an increase in degradation in SERCA proteins with diabetes. In VSMCs from diabetic animals, SERCA2 shifted from a generalized SR distribution to a perinuclear location coupled with an overall decrease in the SERCA protein levels. SERCA2 is the primary Ca
2+-ATPase of the SR in VSMCs and is thought to be most important in cytoplasmic Ca
2+ buffering [
37]. Changes in the thapsigargin-induced Ca
2+ transient suggest that the decrease in protein levels affected the ability of the cells to buffer Ca
2+ via SERCA as has been shown in other labs [
38].
The function of SERCA3 in VSMCs still remains elusive. Originally SERCA3 was reported to be absent from muscle cells. However, thorough examination of tissues with subtype-specific antibodies, revealed that at least one member of the SERCA3 family was equally expressed in all tissues tested [
39]. In human vascular endothelial cells, chronic stimulation with histamine caused an upregulation in the SERCA3 protein, which allowed quicker sequestration of Ca
2+ from the cytoplasm following stimulation [
40]. Given such results in endothelial cells, it is reasonable to consider that a decrease in the SERCA3 levels could partially explain the delayed and blunted response to thapsigargin in the VSMC shown here. The importance of this little defined protein should not be overlooked, as the SERCA3 locus has been implicated in the genetic susceptibility in human type 2 diabetes [
41].
Vascular reactivity to vasopressin in diabetes varies between vascular beds [
42,
43]. For this reason, the protein levels and Ca
2+ responses were tested in VSMCs from both the aorta and the femoral arteries. The diabetes-induced changes in protein levels and in the Ca
2+ responses were the same in VSMCs from either location with the exception of SERCA2 levels, which were increased in the femoral artery, but decreased in the aortic VSMCs in the STZ rat. This increase in the femoral artery agrees with a study finding increased SERCA activity and levels in resistance arteries from diabetic dyslipidemic pigs [
44]. SERCA2 may be of more functional importance in the resistance arteries so these findings may serve as a cautionary note for researchers drawing conclusions from aortic tissue alone.
When analyzing the Ca
2+ channel activity, the results presented here build on earlier work by Ma et al., illustrating a decline in IP
3 responses in VSMCs from STZ treated rats [
28]. The Ma paper showed a decline in vessel contractility and in intracellular Ca
2+ responses via IP
3 receptors and Ca
2+ influx across the plasma membrane. A decrease in IP
3R-1 levels in diabetic arteriolar SMCs has been published previously [
45]. The current study focused the question on the level of specific Ca
2+ regulatory proteins on the SR by conducting live cell experiments in zero extracellular Ca
2+ conditions. Further, we showed that the findings were not dependent on the mode used to induce diabetes, using either a toxin-induced rat model or genetically-bred animals, as the results were generally consistent in the aorta and femoral arteries of the STZ and DR-BB animals. The results suggest that the decreased activity in the VSMCs associated with diabetes is due to shared changes in the SR Ca
2+ release channels (IP
3R) and SERCA resulting in diminished Ca
2+ transients, especially in the nuclear compartment. In addition to selectively activating genes, nuclear Ca
2+ may also play an important role in stabilizing mRNA in a variety of cell types [
46]. The functional impact on differential gene expression of decreased Ca
2+ responses in VSMCs as a result of diabetes is a question that should be intensely pursued, given the findings of this study.
The similarity of the results from identical experiments conducted in freshly dispersed cells from diabetic and control rats, and cultured rat aortic cells grown in normal and high glucose are striking and underlie the importance of chronic exposure to glucose as a major inducer of changes in intracellular Ca
2+ regulation. Previous studies have shown direct effects of glucose on cultured cells, but have typically worked with primary cultures [
47,
48,
35]. In such cases 20-25 mM Ca
2+ in the extracellular media was sufficient to elicit a response similar to that seen in cells from diabetic animals. Due to the fact that we utilized a cultured cell line in which the normal growth media contained 25 mM glucose (referred to as medium in this study), we had to increase the glucose concentration dramatically to evoke a diabetes-like response. While the sensitivity to glucose was different in the culture cells, the other characteristics of the glucose-induced change in freshly dispersed and cultured cells were exceedingly similar.
The importance of separating the nuclear Ca
2+ response from cytoplasmic may not be immediately clear. We are not the first to report on selective Ca
2+ signals in the nuclear that were unique from the cytoplasm as Bkaily et al demonstrated in neurons [
49,
50]. Nuclear and cytosolic Ca
2+ have been reported to be regulated independently in several cell types [
51,
52]. Zones of Ca
2+ signaling around and within the nucleus of smooth muscle cells have led some to suggest that clusters of lysosomes with high densities of RyR form in the perinuclear region forming physical junctions with the SR to comprise a trigger zone of Ca
2+[
53]. The result of the depressed nuclear Ca
2+ signal with high glucose is unknown, but it is likely that VSMCs have nuclear-specific Ca
2+ transcription regulated pathways just as neurons have been shown to possess [
54,
55].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YS carried out the confocal experiments and protein analysis as partial completion of her dissertation requirements. IS oversaw all immunoblot experiments and their analysis. RL was responsible for all animal work and participated in the design of the experiments using the DR-BB and STZ rats. LSB oversaw all work and wrote the manuscript. All authors read and approved the final version of the manuscript.