Syndecans and perlecan are major HSPGs synthesized and secreted by ECs. Syndecans are mainly expressed on the cell surface and perlecan is presented in the ECM . Heparan sulfate chains derived from HSPG are the main GAGs found in ECs . In the present study, we found that total HS determined by HPLC correlated well with total GAGs determined by the carbazole assay (Figure 7). Therefore, total GAGs likely reflect total HS present within endothelium. The GAGs were extracted from the entire cultured endothelial monolayer including mainly syndecans and perlecan.
Total GAGs from the cells and medium were determined when cultured ECs were exposed to high glucose and/or insulin. At the earliest observed time (24 hours), cells treated with high glucose showed a reduction in total GAGs (Figure 1). The observation that cell total GAGs decreased and medium GAGs increased up to 72 hours with high glucose treatment (Figure 3, 4, and 5) are consistent with the finding that GAG contents from syndecan-1 and perlecan were decreased in cultured primary ECs treated with high glucose for two and five days [25, 26]. Decreased cell GAGs suggest hyperglycemia could induce HSPG degradation or inhibit HSPG synthesis in ECs. The possible consequences of decreased GAGs are reduced interaction of several biofactors such as growth factors, coagulation factors, chemokines, adhesion molecules, and lipoprotein lipase with HSPG in the vasculature . Loss of these biofactors is likely to induce endothelial injury. We previously observed endothelial injury following addition of high glucose in the same culture model . Although our culture model uses aortic ECs, and ECs in macro- and micro-vessels have different properties, both are characterized by the same pathological features in diabetes mellitus. Endothelial injury likely contributes to diabetic cardiovascular complications both in micro- and macro-vessels such as nephropathy, retinopathy and atherosclerosis [28, 29].
Increased total GAGs in medium with high glucose treatment suggest that GAGs are released from cell proteoglycan core proteins. The core protein may remain on the cell surface. This was indirectly confirmed by immunoprecipitation assays where the core protein of syndecan-1 remained on ECs and was expressed at lower intensity in medium from high glucose treated ECs . Similarly, immunostaining studies showed that reduction in HS GAGs in the GBM under diabetic conditions was not accompanied by a reduction in the HSPG core protein . As well, decreased HSPG synthesis by human aortic ECs in high glucose conditions was not the result of a decrease in GAG size, further suggesting that entire GAG chains were released into culture medium . It is unlikely that GAGs are further degraded in medium.
Insulin controls blood glucose utilization and influences the metabolism of fat and protein. Insulin also has effects on the expression of numerous genes. Insulin alone decreased GAG concentration in cells at all time points in our study. In a study of regulation of HSPG metabolism and hepatocyte growth by insulin and phosphatidylinositol, insulin markedly stimulated the rate of internalization of matrix HSPG and phospholipase C and therefore may control cell surface HSPG turnover . Thus insulin may regulate enzymes involved in metabolism of proteoglycans. Insulin promoted shedding of syndecan ectodomains, from 3T3-L1 adipocytes, by an unknown mechanism . However, it is unknown whether these changes are coordinated by phosphatidylinositol, the second messenger in the action of binding insulin to its receptor, and whether shedding of syndecans are seen in ECs in response to insulin. In our studies there was a trend that insulin alone increased GAGs in culture medium as culturing time increased (Figure 4) which may indicate HSPG turnover or shedding of syndecan into medium, however, GAGs were significantly reduced in 72 hour cultures compared to control suggesting that GAG synthesis may also be inhibited. These limited observations cannot define the precise mechanisms involved in insulin reduction of GAGs in cultured ECs, and further investigation is required. Considerable evidence indicates that vascular endothelium is a physiological target of insulin and a potential link between insulin resistance and atherosclerosis [33, 34]. Endothelial dysfunction is one of the earliest detectable signs in insulin resistance . Our present study shows that decreased GAG content in insulin alone treated cells may lead to endothelial dysfunction caused by GAG degradation and/or inhibition of GAG synthesis.
The evidence for insulin influencing different cultured cells under high glucose conditions is varied and controversial. Studies on cultured mesangial cells treated with glucose and insulin showed insulin did not influence HSPG content independent of the ambient glucose levels . Insulin was unable to correct the 30 mM glucose induced reduction in HSPG synthesis of rat glomerular epithelial cell layers that resemble the GBM . However, reduction in chondroitin sulfate proteoglycan synthesis found in articular cartilage in diabetic rats could be completely restored by administration of insulin . Administration of large doses of insulin restored HSPG synthesis in basement membrane following reduction of HSPG by implantation of Engelbreth-Holm-Swarm (EHS) tumor cells in diabetic mice . Our current studies showed that: insulin increased GAGs in high glucose treated cells compared to insulin alone at all times and to high glucose alone at 48 and 72 hours; insulin increased medium GAGs at all times compared to control, at 24 and 72 hours compared to insulin alone; insulin was able to maintain high glucose treated cell+medium GAGs at control levels at 72 hours (Figure 4C). Previous studies noted that insulin alone increased endothelin-1 (ET-1) levels . Perhaps insulin maintains HSPGs under hyperglycemic conditions through its effects on ET-1. Although these studies indicate that insulin has the potential to improve hyperglycemia induced alteration of HSPG in ECs, the effect is time dependent. It is possible that insulin modulation of proteoglycan metabolism under hyperglycemic conditions is cell or tissue specific and specific for individual species of proteoglycans.
Heparin used as an antithrombotic drug, is also considered a potent vasodilator , and lowers blood pressure . Heparin has the ability to inhibit heparanase upregulation induced by high glucose in cultured ECs . Our results suggest that heparin increases GAG content in cells treated with high glucose suggesting the protective effect of heparin on ECs. Taken together with evidence that heparin stimulates HS synthesis and modification of HS in ECs [43, 44], further suggests that heparin's protective action may be due to an increase in GAG synthesis or inhibition of heparanase production.