Cardiac Microvascular Pathology In Early Diabetic Rat And The Relationship With VEGF And PKCβⅡ

【Background】It is estimated that there are more than 100 million people with the diagnosis of diabetes mellitus (DM) in the worldwide. As far as heart disease concerned, multiple lines of evidences support the viewpoint that diabetes mellitus is an independent risk factor for cardiovascular disease and diabetes is associated with a 2- to 4-fold increase in the risk of developing coronary artery disease. At the same moment, there is an ever-increasing body of evidence that indicates that cardiac microvessel makes great contributions primarily to diabetic heart disease. In early stage of diabetes, there exists well-recognized tissue ischemia for the vascular dysfunction of relaxation/constriction and permeability. And the collateral circulation in heart is induced by some factors to improve cardiac blood supply. However, more severe cardiac complications are associated with patient in diabetes.Owing to little obvious clinical symptom found in early diabetes, previous studies focused their attentions on midanaphase (3-12 months after diabetic model establisment) and through the quantity/function changes of cardiac vascular endothelium to describe microvascular abnormalities. Notwithstanding, the direct observation on cardiac microvascular pathology in early diabetes (1-2 months after diabetic model establishment) has not been obtained.Furthermore, great efforts have been made to explain the relationship between VEGF and cardiac microvessels in diabetes for its significant role in vascular angiogenesis and the development of cardiac vascular complications in diabetes. However, there emerges dissention with the expression pattern of VEGF and VEGF receptors, which is addressed with a paramount role on the aspects correlated with vascular endothelia cell. The variance in the phenomenon still remains to be elucidated.Activity of the protein kinase C (PKC) family, especially PKC–βⅡ, has been well documented in diabetic myocardium for its numerous biological responses. However, little attention is addressed on PKC-βⅡand its inhibitor LY333531 in cardiac microvascular permeability dysfunction, which takes a significant role in the pathophysiological cascade of diabetic heart disease.【Objectives】1. To evaluate cardiac microvascular pathology in early diabetes and the relationship with VEGF and VEGFR2;2. To observe cardiac PKCβⅡexpression in vivo in early diabetes and investigate the antagonism of LY333531 on the increased permeability of cardiac microvascular endothelial cells (CMECs) induced by high glucose in vitro.【Methods】Part 1:1. Model establishment: male Sprague Dawley (SD) rats (6 weeks) were raised with special food for 8 weeks. After STZ peritoneal injection (50mg/kg) and usual breeding for 4 weeks, the early diabetic model was established if random blood glucose≥16.7mmol/L. Age-matched normal rats were included in control group;2. After retrograde cannulation through right carotid artery to left vetircle, hemodynamic properties of rats were recorded with computer;3. Using vascular cast mould, cardiac microvascular angiogenesis was observed under scaning electron microscope;4. With lanthanum as tracer, cardiac microvascular permeability observations were performed under transmission electron microscope;5. Immunofluorescence for vascular endothelium cell count and TUNEL for apoptosis were taken;6. The expression patterns of VEGF and VEGFR2 between groups were compared by histopathologic analysis and Western blot.Part 2:1. Same with step 1 of part 1;2. The expression of PKCβⅡwas compared between normal and DM heart sample through immunofluorescence; In vitro experiment:3. After tissue separation, CMECs were cultured according to different rates of attachment and identificated by AcLDL staining;4. The cultured CMECs were randomly divided into four groups: normal group, high glucose group (25mmol/L), high glucose+LY333531 (10nmol/L) group and high glucose+saline group;5. The permeability of cell monolayer was detected using in vitro vascular permeability assay kit; 6. Cell apoptosis was determined by TUNEL;7. The expression of PKCβⅡin vitro was analyzed by immunofluorescence and Western blot in each group.【Results】Part 1:1. 25 early DM models were established and the achievement ratio was more than 80%; Compared with normal group,2. There appeared diastolic and systolic dysfunction in the models of early DM;3. Pathological cardiovascular angiogenesis with irregular styles was clearly defined in early DM;4. Increased microvascular permeability is obvious in early DM group,5. Decreased endolthelial cells and increased apoptosis in DM heart is also significant enough (P<0.01);6. The up-regulation of VEGF and down-regulation of VEGFR2 in early DM group is performed significantly (P<0.01).Part 2:1. The condition of model establishment is same with Part 1;2. Compared with normal group,increased cardiac expression of PKCβⅡin early DM was demonstrated obviously in vivo. In vitro experiment:3. According to the result of AcLDL staining, CMECs were proved to be cultured successfully.4. Compared with normal group, the permeability of cell monolayer cultured in high glucose medium was increased (P<0.01) at a higher apoptosis rate (P<0.01) and PKCβⅡexpression (P<0.01).5. The high glucose + LY333531 group showed noticeable attenuation on both permeability (P<0.01) and apoptosis (P<0.01) with reduced PKCβⅡexpression (P<0.01). No significant effects of saline on the cell permeability, apoptosis and PKCβⅡexpression were observed.【Conclusions】1. The cardiac microvascular pathology in early DM is described with the microvascular dysfunction of permeability and angiogenesis.2. The pathological state may be accelerated and aggravated by the disbalance between up-regulation of VEGF and down-regulation of VEGFR2.3. The effect of high glucose is enough to induce the impairment of permeability of CMECs, which is well correlated with the role of PKCβⅡ4. As PKCβinhibitor, LY333531 is competent to attenuate the permeability dysfunction induced by high glucose.