| Background:Calcium sensing receptor (CaSR, also known as GPRC2A) is a member of family C in G protein coupled receptors (GPCRs). In1993, CaSR was firstly cloned from bovine parathyroid cells and defined as an important modulator of parathyroid hormone (PTH) secretion through sensing extracellular calcium fluctuation. Initially it is reported that CaSR is highly expressed in tissues maintaining systemic calcium homeostasis, such as parathyroid gland, thyroid gland, kidney, bone and gastrointestinal tract. Nevertheless, functional CaSR is indentified in more and more tissues including brain, heart, aorta, esophagus, stomach, pancreas, ovary, testis and so on. Extensive expression of CaSR reveals that the biological function of CaSR is not limited in sensing calcium ions oscillation and maintaining systemic calcium homeostasis. CaSR was demonstrated modulating multiple physiological process and basic cell activities (e.g. cell proliferation, differentiation, chemotaxis, survival and apoptosis). Recently, full-length of CaSR has been detected in cardiovascular system and manifested as functional regulator in many physiological and pathological processes, such as vasoconstriction and myocardial ischemia-reperfusion. However, the role of CaSR in various evoked vascular lesions (e. g. aging induced vascular pathological changes, diabetic vascular disease, and COPD induced atherosclerosis) is poorly discussed. According to existing research results, we promote a hypothesis that CaSR is involved in initiation and development of diabetic macroangiopathy.Objectives:1. To explore the molecular pathological changes in diabetic rats thoracic aorta2. To reveal the transcription and translation pattern of CaSR in normal and DM rats thoracic aorta, and then explore the possible relationship between CaSR and diabetic macroangiopathy. 3. To detect the transcription and translation pattern of CaSR in both normal and hyperglycemia induced apoptotic endothelial cell.4. To discover the effect of CaSR modulator (NPS-2143) on hyperglycemia induced apoptotic endothelial cell as well as possible molecular mechanism and signaling pathway.Methods:Experiments in viva1. Rat diabetic model was induced via high-fat diet feeding and streptozotocin injection, and the hematoxylin-eosin staining of aorta paraffin section was conducted to show the pathological changes of aorta thoracica in model rats. At the same time, levels of non-esterified fatty acid, trigryceride, total cholesterol, endothelin-1and nitric oxide in serum as well as levels of hydroxyproline, collagen I, collagen III, endothelin-1, nitric oxide, cell apoptosis, cleaved caspase-3and caspase-9in vessel wall, which showed as molecular markers of vascular fibrosis and endothelial dysfunction, were determined via serum biochemical assay, tissue biochemical assay, real time PCR, TUNEL staining and western blotting.2. Expression pattern of CaSR was detected in control and model aorta via real time PCR and western blotting.3. Phosphorylation level of JNK and p-38, which acted as apoptosis related kinases downstream of CaSR, and protein level of pro-apoptotic Bax were assayed by western blotting.Experiments in vitro.1. Human umbilical vein endothelial cell line (HUVEC) was stimulated with high concentration of glucose or glucosamine to mimic hyperglycemia in vitro. Then, the effect of stimulus and CaSR regulator on endothelial cell viability and caspase activation was determined via CCK-8method as well as western blotting.2. The effect of stimulus and CaSR inhibitor on the level of endothelin-1as well as nitric oxide was detected by cell based biochemical assay. The variation tendency of CaSR expression, JNK/p-38phosphorylation and Bax expression caused by stimulus and CaSR modulator administration was researched via real time PCR and western blotting.Results:Diabetes animal model related assays demonstrated that:1. Visible pathological changes of aorta in diabetic rats could not be found as shown in HE staining results.2. Compared with control group, serum non-esterified fatty acid, triglyceride and total cholesterol levels were obviously elevated in diabetic rats (P<0.01). Increasing of serum and intracellular endothelin-1level as well as decreasing of serum and intracellular nitric oxide level were observed in model group (P<0.01). The up-regulation of hydroxyproline, collagen I and collagen Ⅲ was found in the aorta thoracica of diabetic rats (P<0.05). And then, the significant promotion of TUNEL-positive apoptotic cells and cleaved caspase were uncovered in diabetic aorta thoracica (P<0.05).3. CaSR transcription and expression were significantly up-regulated in the hyperglycemia model group (P<0.005). Pro-apoptotic protein Bax and apoptosis initiating kinase-JNK and p-38were enhanced in model group accordingly (P<0.01).Cell-based measurements demonstrated that:1. High concentration of glucose and glucosamine stimulation could induce cell apoptosis, and cell viability of stimulated groups decreased50%and70%respectively (P<0.005). Nevertheless, treatment of CaSR specific inhibitor NPS-2143could partially restore cell viability impaired by stimulus (P<0.005).2. Intracellular endothelin-1level in stimulated group increased to140%of that in low glucose control group and intracellular nitric oxide level in stimulated group decreased to80%of that in control group (P<0.05), while intracellular endothelin-1level in NPS-2143and stimulus co-treatment group increased to105%of that in stimulated group and intracellular nitric oxide level in NPS-2143and stimulus co-treatment group only showed decreasing tendency without statistical significance (p=0.586). 3. CaSR level and key apoptosis-involved proteins (cleaved caspase-3,-9, Bax, phospho-p38and phosphor-JNK) were elevated in response to stimulation (P<0.01) and inhibition of CaSR by using specific inhibitor NPS-2143could reverse the elevation induced by stimulus (P<0.01).Conclusions:CaSR play an important role in development of diabetic angiopathy via promoting apoptosis of endothelial cell induced by hypercemia. Our study indicates the mechanism is through mobilizing JNK/p-38-Bax-caspase-9-caspase-3pathway, while CaSR inhibitor (NPS-2143) could protect endothelial cells from hyperglycemia induced apoptosis by suppressing JNK/p38-Bax pathway in cell-based assays. |
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