| BackgroundAmount of researchers showed interest in close relationships between diabetes and cardiovascular disease. If diabetes patients received revascularization, accidence related with reperfusion often happened. These indicated diabetic heart is more sensitive to ischemic reperfusion injury than the nondiabetic heart. This phenomenon was confirmed by cardiacmyocyte death increase, severe impaired heart function and incidence of no-reflow rose. Cardiac microvascular barrier dysfunction, commonly occurring before microvascular sclerosis, is considered to be one of the initiating mechanisms that underlie the pathogenesis of diabetes microvascular complications. These phenomenons indicate endothelial barrier dysfunction probably significantly contributes to subsequent functional and cellular injury during I/R through a variety of pathological pathways and I/R in diabetes extending into the microvasculature probably is more severe.Early impairment of microvascular complication of diabetes was deterioration of cell junction and cell barrier function. This pathologic change may increase paracellular flux of injury factor, which could worse inflammatory reaction, aggravate apoptosis of endothelium and deteriorate microvascular function. Above pathology phenomena wildly existed in microangiopathy-prone tissues, such as the retina, renal glomeruli and heart. PKC was activated, which is strongly implicated in the pathogenesis of diabetic microangiopathy. PKC-βII isoforms exhibit a greater increase in the membrane fractions of many vascular tissues than the other isoforms in diabetes. Ruboxistaurin(Rx), a high selective PKC-βinhibitor, make the study about the role of PKC activation in microvascular complication more deeply. However, research about the role of PKC-βII activation in cardiac microvascular barrier function was seldom and the mechanism didn't confirm. It has not been previously established whether PKC-βII activation play important role in I/R injury of cardiac microvessels in diabetes. Under this condition, this study paid attention to the role of PKC-βII activation in cardiac microvascular barrier function and ischemia reperfusion injury in cardiac micvasculture on diabetic rats.AIM1. Diabetic rat model was established and to determine whether PKC-βII activation existed in heart tissue of diabetic rats. Pathologic relationship between cardiac microvascular barrier dysfunction and PKC-βII activation was explored;2. The model of diabetic rat subjected to I/R was established and to observe characters of cardiac microvascular impairment in diabetic rat. In addition, to demonstrate whether this effect could be prevented by a PKC-βinhibitor and to prove PKC-βII activation involved in the mechanism by which CMECs were impaired during I/R.3. To observe permeability of CMECs monolayer in high glucose environment and the mechanism of PKC-βII activation influence barrier function.4. To elucidate whether barrier function aggravated in CMECs monolayer cultured in high glucose medium subjected to SI/R. To proved PKC-βII activation involved in the mechanism by which CMECs impairment worse during I/R.Methods1. Male Sprague-Dawley rat, 120-150 g, dieted with high glucose and high cholesterol for 8 weeks, diabetes was induced with a single intraperitoneal injection of streptozotocin (50 mg/kg, Sigma). Random serum glucose greater than 16.7mmol/L were considered success.2. Diabetic rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.), and experienced left thoracotomy. And established I/R experimental models. Rats were subjected to 30 min myocardial ischemia and 72 h reperfusion.3. Cardiac microvascular permeability and ultrastructure were compared with Lanthanum as a tracer. Casting of cardiac microvessels perfused by Mercox observed under SEM.4. PKC-βII location and VE-cadherin rearrangement in diabetic heart tissue were examined by immunofluorescence. Phosphorylated LIM kinase 2 in heart tissue after I/R was determined by immunohistological method.5.±LVdp/dtmax of after I/R and±LVdp/dtmax of before I/R were recorded by 8–orbits physiological grapher.6. Thioflavin S was injected into the heart to define the region of no-reflow. Thioflavin S is used as a standard marker for identifying zones of no-reflow and to calculated no-reflow area.7. After removal of the endocardial endothelium and the epicardial coronaries, the left ventricles were cut into small pieces and incubated in 2 ml 0.2% collagens, isolated and purified .Cardiac microvascular endothelium cells(CMECs).8. Quantitatively assess permeability of CMECs monolayer using In Vitro Vascular Permeability Assay kit.9. PKC-βII, VE-cadherin, phospho-β-catenin and Phosphorylated LIMK2 was determined by western blot analysis.10. TUNEL staining detected apoptosis of CMECs in diabetic rat and in high glucose medium subjected to I/R. Three staining of CD31, TUNUL and DAPI were used to obtain AI of CMECs of diabetic rats after I/R.11. F-actin was stained with FITC-phalloidin and cells were visualized with fluorescence microscopy.12.G-actin / F-actin assay kit was used to quantitatively detected F-actin.Results1. PKC-βII activation play a key role in microvascular barrier dysfunction on diabetic rat's heart.(1) Successfully established diabetic rat model with FPG increase.(2) IF examined PKC-βII activation and VE-cadherin rearrangement in diabetic rats'heart. PKC-βII was downregulated and VE-cadherin expression was regular after Rx treatment(3) Relative expression of PKC-βII (1.06±0.09 vs. 0.76±0.06,P<0.01)and P-β-catenin(0.74±0.13 vs.0.47±0.11, P<0.01)in diabetic rats increased compared with that in non-diabetic control. PKC-βII expression (( 0.79±0.10 vs. 0.97±0.11,P<0.05) and P-β-catenin (0.59±0.07 vs. 0.80±0.11, P<0.05) were significantly downregulated by pretreatment with Rx.(4) Lanthanum as a tracer to found cariac microvascular dysfunction in diabetic rat and the surfaces of cardiac microvascular casting weren't integrity inder electron microscope. Rx administration could attenuate this disturbance.2. PKC-βII activation had important role in cardiac microvascular function impairment aggravated in diabetic rat during I/R. (1)±LVdp/dt decreased in DM group compared with non-diabetic control and sham, and Rx administration could improve±LVdp/dt in DM group.(2) No-reflow size in diabetic rats significantly larger than sham (20.0±1.7% vs. 0%) and non-diabetic control (20.0±1.7% vs.14.3±1.8%, P<0.01), Treatment with Rx significantly reduced the no-reflow size (16.4±1.9% vs. 19.4±1.6%, P<0.05).(3)CMECs were severely impaired after I/R in diabetic rats. The edema and vacuolus were represented in diabetic rats. Rx pretreatment could attenuate this severe pathology.(4)AI of CMECs in diabetic heart increased compared with that in sham (12.6±2.2% vs.8.1±1.7%, P<0.01)and non-diabetic control(12.6±2.2% vs.7.8±0.8%, P<0.01). AI of CMECs in Rx treated group reduced(9.5±1.5% vs. 12.2±2.1%, P<0.05).(5)Relative expression of Phospho-LIMK2 increased compared with that in Sham (1.01±0.10 vs. 0.66±0.11, P<0.01)and non-diabetic control(1.01±0.10 vs. 0.60±0.09, P<0.01 ) . Phospho -LIMK2 expression was significantly downregulated by Rx administration(0.76±0.08 vs. 0.97±0.10, P<0.01).3. PKC-βII activation decrease the barrier function of CMECs monolayer in High glucose medium.(1) Permeability of monolayer cell in high glucose medium was higher than in normal medium (394.50±36.92 vs. 282.10±14.93, P<0.01).However, the addition of PKC-βII i?nhibitor, Rx (1, 10, 100nmol/L), ?resulted in permeability decreased (P<0.01).(2) PKC-βII (1.17±0.13 vs.0.71±0.07, P<0.01)and phospho-β-catenin(0.92±0.11 vs. 0.56±0.07, P<0.01) expression in CMECs incubated with high glucose medium increased compared with that in normal medium. PKC-βII and phospho-β-catenin expression was significantly down regulated given Rx (10nmol/L) pretreatment (P<0.01). VE-cadherin(c-19)didn't alter in CMECs cultured in high glucose medium.4. PKC-βII activation was involved in the mechanism by which CMECs impairment worse during I/R.(1) Permeability of monolayer cell in high glucose medium was higher than in sham (590.40±34.38 vs. 359.20±22.80, P<0.01) and in normal medium (590.40±34.38 vs. 383.70±37.58 P<0.01).However, the addition of Rx (10nmol/L) could resulte in permeability decreased (432.00±30.83 vs. 571.10±32.60P<0.01).(2) Phospho -LIMK2 of CMECs in high glucose medium increased compared with that in sham (1.11±0.09vs. 0.31±0.09, P<0.01)and in normal medi( 1.11±0.09vs. 0.43±0.15, P<0.01 ) . Phospho -LIMK2 expression was significantly downregulated by pretreatment with Rx ( 0.60±0.08 vs.1.28±0.11,P<0.01).(3) AI of CMECs in high glucose medium subjected to I/R increased compared with that in sham (13.3±1.3% vs. 5.7±1.4%, P<0.01)and in normal medium (13.3±1.3% vs. 7.8±0.8%, P<0.01). AI could be decreased by pretreatment with Rx(9.1±1.1% vs.12.8±1.8%, P<0.01).(4)The ratios of F-actin to G-actin in CMECs incubated with high glucose medium increased compared with that in sham (10.73±1.32 vs. 6.58±1.34, P<0.01)and in normal medium (10.73±1.32 vs. 7.73±0.97, P<0.01).Pretreatment with Rx (10nmol/L) could reduce it.(7.32±1.02 vs.10.24±1.38, P<0.05).Conclusion1. STZ induced diabetic rat model was used to verify that diabetic state induced activation of PKC-βII and microvascular complication displaying in permeability increase or microvascular barrier dysfunction. Phospho-β-catenin expression and VE-cadherin rearrangement involved in this mechanism; 2. Exaggerated no-reflow areas accompanying with severe impairment of cardiac function and aberrant microvascular barrier function presented in diabetic rat model after I/R and this effect could be prevented by a PKC-βinhibitor pretreatment. PKC-βII-dependent Rho kinase activation in diabetes in response to I/R and phosphor- LIMK2 increase contributed to microvascular damage and the severe no-reflow phenomenon;3. High glucose microenvironment could activate PKC-βII in cultured CMECs and lead to phospho-β-catenin increase. It promoted the dissociation of VE-cadherin–catenin complex and deteriorated the CMECs barrier function;4. PKC-βII was activated in CMECs cultured in high glucose, and in this state subjected to I/R, Rho kinase was excessively activated and phospho-LIMK2 increased. Phospho-LIMK2 was involved in structural alterations in cytoskeletal filaments and polymerization of F-actin which formed concentrical force. Loss of equilibrium in VE-cadherin and F-actin could aggravate CMECs impairment after I/R.These results indicated pretreatment for diabetic microvascular complication induced by PKC-βII could be helpful to attenuate the risk of I/R injury.
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