New paeonol derivative C302 reduces hypertension in spontaneously hypertensive rats through endothelium-dependent and endothelium-independent vasodilation.

Hypertension is a major risk factor for cardiovascular disease and Chinese herb monomers could provide new structural skeletons for anti-hypertension new drug development. Paeonol is a Chinese herbal monomer extracted from Cortex moutan, exhibited some anti-hypertensive activity. The study focused on the structural optimization of paeonol to provide promising lead compounds for anti-hypertension new drug development. Herein, twelve new paeonol derivatives (PD) were designed and synthesized and their vasodilation activity was evaluated by in vitro vasodilation drug screening platform based on Myograph. Its anti-hypertension activity, PD-C302 (2-hydroxy-4-methoxyvalerophenone) as a representative with the optimal vasodilation activity, was determined by its response to blood pressure in spontaneously hypertensive rats (SHR) in vivo. Moreover, its molecular mechanism was probed by the vasodilation activity of rat superior mesenteric artery rings with or without endothelium pre-contracted by potassium chloride (KCl) or phenylephrine hydrochloride (PE). It was indicated that PD-C302 significantly reduced the blood pressure in SHR, which would involve in PD-C302-induced vasodilation. Furthermore, endothelium-dependent pathways and endothelium-independent pathways both contributed importantly to PD-C302-induced vasodilation at low concentration of PD-C302. Endothelium-independent pathways (vascular smooth muscle cell-mediated vasodilation), were mainly responsible for the PD-C302-induced vasodilation at high concentration of PD-C302, which involved in opening multiple K+ channels to restrain Ca2+ channels, and then triggered vasodilation to reduce blood pressure. PD-C302 has a simple structure and favorable anti-hypertensive activity in vivo, which could be a promising lead compound for anti-hypertension new drug development.

The OPG/RANKL/RANK system modulates calcification of common carotid artery in simulated microgravity rats by regulating NF-κB pathway.

Functional and structural adaptation of common carotid artery could be one of the important causes of postflight orthostatic intolerance after microgravity exposure, the mechanisms of which remain unclear. Recent evidence indicates that long-term spaceflight increases carotid artery stiffness, which might present a high risk to astronaut health and postflight working ability. Studies have suggested that vascular calcification is a common pathological change in cardiovascular diseases that is mainly manifested as an increase in vascular stiffness. Therefore, this study investigated whether simulated microgravity induces calcification of common carotid artery and to elucidate the underlying mechanisms. Four-week-old hindlimb-unweighted (HU) rats were used to simulate the deconditioning effects of microgravity on cardiovascular system. We found that simulated microgravity induced vascular smooth muscle cell (VSMC) osteogenic differentiation and medial calcification, increased receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and RANK expression, and enhanced NF-κB activation in rat common carotid artery. In vitro activation of the RANK pathway with exogenous RANKL, a RANK ligand, increased RANK and osteoprotegerin (OPG) expression in HU rats. Moreover, the expression of osteogenic markers and activation of NF-κB in HU rats were further enhanced by exogenous RANKL but suppressed by the RANK inhibitor osteoprotegerin fusion protein (OPG-Fc). These results indicated that the OPG/RANKL/RANK system modulates VSMC osteogenic differentiation and medial calcification of common carotid artery in simulated microgravity rats by regulating the NF-kB pathway.

[The molecular mechanism of fibroblast growth factor 21-inhibited leptin expression in adipocytes].

The present study was aimed to clarify the signaling molecular mechanism by which fibroblast growth factor 21 (FGF21) regulates leptin gene expression in adipocytes. Differentiated 3T3-F442A adipocytes were used as study object. The mRNA expression level of leptin was detected by fluorescence quantitative RT-PCR. The phosphorylation levels of proteins of signal transduction pathways were detected by Western blot. The results showed that FGF21 significantly down-regulated the mRNA expression level of leptin in adipocytes, and FGF21 receptor inhibitor BGJ-398 could completely block this effect. FGF21 up-regulated the phosphorylation levels of ERK1/2 and AMPK in adipocytes. Either ERK1/2 inhibitor SCH772984 or AMPK inhibitor Compound C could partially block the inhibitory effect of FGF21, and the combined application of these two inhibitors completely blocked the effect of FGF21. Neither PI3K inhibitor LY294002 nor Akt inhibitor AZD5363 affected the inhibitory effect of FGF21 on leptin gene expression. These results suggest that FGF21 may inhibit leptin gene expression by activating ERK1/2 and AMPK signaling pathways in adipocytes.

The GPR120 Agonist TUG-891 Inhibits the Motility and Phagocytosis of Mouse Alveolar Macrophages.

Movement and phagocytosis characterize the fundamental actions of macrophages. Although it is known that the free fatty acid receptor GPR120 is expressed in macrophages and regulates cytokine expression to exert anti-inflammatory activities, the effects of GPR120 activation on the motility and phagocytosis of macrophages are not clear. In this study, mouse alveolar macrophages (AM) were stimulated with the GPR120 agonist TUG-891, and the changes in cell motility, intracellular Ca2+ concentration ([Ca2+]i), and the ability of phagocytosis were measured. Mouse AM in controls exhibited active movement in vitro, and TUG-891 significantly restrained AM movement. Meanwhile, TUG-891 stimulated a quick increase in [Ca2+]i in AM, which was blocked separately by the Gq protein inhibitor YM-254890, the phospholipase C (PLC) inhibitor U73122, or depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin. The inhibition of AM movement by TUG-891 was eliminated by YM-254890, U73122, thapsigargin, and chelation of cytosolic Ca2+ by BAPTA. Moreover, TUG-891 inhibited AM phagocytosis of fluorescent microspheres, which was also blocked by YM-254890, U73122, thapsigargin, and BAPTA. In conclusion, GPR120 activation in mouse AM increases [Ca2+]i but inhibits the motility and phagocytosis via Gq protein/PLC-mediated Ca2+ release from ER Ca2+ store.

Mitochondrial-Derived Peptide MOTS-c Attenuates Vascular Calcification and Secondary Myocardial Remodeling via Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

INTRODUCTION: Vascular calcification (VC) is a complex, regulated process involved in many disease entities. So far, there are no treatments to reverse it. Exploring novel strategies to prevent VC is important and necessary for VC-related disease intervention. OBJECTIVE: In this study, we evaluated whether MOTS-c, a novel mitochondria-related 16-aa peptide, can reduce vitamin D3 and nicotine-induced VC in rats. METHODS: Vitamin D3 plus nicotine-treated rats were injected with MOTS-c at a dose of 5 mg/kg once a day for 4 weeks. Blood pressure, heart rate, and body weight were measured, and echocardiography was performed. The expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and the angiotensin II type 1 (AT-1) and endothelin B (ET-B) receptors was determined by Western blot analysis. RESULTS: Our results showed that MOTS-c treatment significantly attenuated VC. Furthermore, we found that the level of phosphorylated AMPK was increased and the expression levels of the AT-1 and ET-B receptors were decreased after MOTS-c treatment. CONCLUSIONS: Our findings provide evidence that MOTS-c may act as an inhibitor of VC by activating the AMPK signaling pathway and suppressing the expression of the AT-1 and ET-B receptors.

Erythropoietin attenuates vascular calcification by inhibiting endoplasmic reticulum stress in rats with chronic kidney disease.

Previous studies suggested that endoplasmic reticulum (ER) stress induced-apoptosis promoted vascular calcification (VC). Interestingly, erythropoietin (EPO), an endogenous glycoprotein, exerts multiple tissue protective effects by inhibiting ER stress and apoptosis. We investigated the role and potential mechanism of EPO on VC in chronic kidney disease (CKD) rats and cultured vascular smooth muscle cells (VSMCs). The calcification model was established by subtotal nephrectomy in vivo or phosphate overload in vitro. The protein level of EPO receptor (EPOR) was increased in the calcified aortas of CKD rats. EPO prevented the reduction of VSMC phenotypic markers, and reversed the increased calcium content and calcium salt deposition in the aortas of CKD rats and cultured calcified VSMCs. The protein levels of activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94) were upregulated in aortas and VSMCs under calcifying conditions, indicating ER stress activation. EPO treatment of CKD rats or calcified VSMCs downregulated the protein levels of ATF4 and GRP94. Furthermore, ER stress-mediated apoptosis, determined by the protein levels of CCAAT/enhancer-binding protein-homologous protein and cleaved caspase 12, was increased in tunicamycin or calcification media-treated VSMCs, but the increased effect was reversed in EPO-treated groups. The increased apoptotic cells in calcified VSMCs, as indicated by Hoechst staining and flow cytometry, were downregulated by the co-administration of EPO or 4-phenyl butyric acid. In conclusion, EPO might attenuate VC by inhibiting ER stress mediated apoptosis through EPOR signaling.

CaMKII mediates myocardial ischemia/reperfusion injury­induced contracture in isolated rat heart.

Contracture or diastolic dysfunction is a primary cause of injury following ischemia/reperfusion (IR). The present study examined whether Ca2+/calmodulin­dependent kinase II (CaMKII) is involved in contracture. Isolated rat hearts were subjected to either global IR or Ca2+ paradox (CaP), which is characterized by contracture. Left ventricular end­diastolic pressure, electron microscopy and troponin I  TnI) in coronary effluent were examined to indicate the extent of contracture. Compared with control hearts, both the IR and CaP groups exhibited an increase in necrosis and apoptosis, and a marked depression in contractile function. Western blot analysis showed that IR stimulated the phosphorylation (Thr287) and oxidation (Met281/282) of CaMKII, and the phosphorylation of phospholamban (PLN), a substrate of CaMKII. By contrast, only the phosphorylation of CaMKII was increased in the CaP group. Treatment with either 3 µM KN­62, an inhibitor of CaMKII, or 5 µM KB­R7943, an inhibitor of the Na+/Ca2+ exchanger, mitigated the damage and the post­translational modification of both CaMKII and PLN. Similar to the effect of the negative inotropic agent 2,3­butanedione­monoxime, the increased cell survival after treatment with KN­62 was associated with improved diastolic function. Examination using electron microscopy and a biochemical test showed the development of contraction bands, disruption of the sarcolemmal membrane and an increase in the release of TnI in both IR and CaP hearts; these results indicated the occurrence of contracture. Furthermore, these changes were inhibited by either KN­62 or KB­R7943. Taken together, these data provided evidence that CaMKII mediates reperfusion­elicited contracture, and that the activation of CaMKII via phosphorylation is involved in this process.

Grifolic acid causes osteosarcoma cell death in vitro and in tumor-bearing mice.

Grifolic acid is a natural compound isolated from the fungus Albatrellus confluens. In the present study, we assessed the effects of grifolic acid on human osteosarcoma cells. We found that grifolic acid dose- and time-dependently induced cell death in the U-2 OS, MG-63, Saos-2, and 143B human osteosarcoma cell lines. Grifolic acid decreased osteosarcoma cell mitochondrial membrane potential, ATP production, and cellular NADH levels, but did not impact mitochondrial membrane potential in isolated mitochondria from human osteosarcoma cells. Intratumoral injection of grifolic acid also promoted tumor cell death and prolonged survival in nude mice bearing human osteosarcoma xenografts. Grifolic acid had no obvious toxicity in mice, with no histological changes in liver, kidney, lung, or heart, and no changes in blood cell counts or levels of plasma total protein, alanine aminotransferase, or aspartate aminotransferase. These results show that grifolic acid induces osteosarcoma cell death by inhibiting NADH generation and ATP production without obvious toxicity. Intratumoral injection of grifolic acid may be a promising anti-osteosarcoma therapeutic option in patients.

[Inhibition of CaMKII alleviates myocardial ischemia?reperfusion injury by reducing mitochondrial oxidative stress in isolated perfused rat heart].

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in myocardial ischemia-reperfusion (IR) injury in isolated perfused rat heart and explore the underlying mechanisms. METHODS: An ischemia-reperfusion (IR) model was prepared using isolated rat hearts perfused with Krebs-Henseleit solution were randomly divided into control group, 2.5 µmol/L KN-93 group, IR (induced by ischemia for 45 min followed by reperfusion for 120 min) group and KN-93+IR group. The myocardial performance was evaluated by assessing the left ventricular pressure. Lactate dehydrogenase (LDH) activity and cTnI content in the coronary flow and the infarct size were determined to evaluate the myocardial injury. The phosphorylation of CaMKII (p-CaMKII) and PLN (p-PLN) and oxidation of CaMKII (ox--CaMKII) were measured with Western blotting. The activity of mitochondrial superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using ELISA. RESULTS: Compared with the control group, KN-93 treatment at 2.5 µmol/L produced no significant effects on cardiac function or performance in rat hearts without IR injury. Myocardial IR injury significantly decreased myocardial performance and mitochondrial SOD activity in the perfused hearts (P<0.01) and caused significantly increased infarct size, LDH activity, cTnI content, expressions of p-CaMKII, ox-CaMKII and p-PLN, and also increased mitochondrial MDA content (P<0.01). KN-93 treatment at 2.5 µmol/L administered before ischemia and before reperfusion markedly attenuated such changes induced by ischemia and reperfusion (P<0.01). CONCLUSION: CaMKII participates in myocardial IR injury in isolated rat heart, and inhibiting CaMKII can alleviate myocardial injury by relieving mitochondrial oxidation stress.

MiR125b-5p protects endothelial cells from apoptosis under oxidative stress.

Endothelial cell damage, such as apoptosis and necrosis, is involved in many cardiovascular diseases. In recent years, the crucial role of microRNAs in controlling tissue homeostasis and disease in the epithelium has become widely recognized. In the present study, human umbilical vein endothelial cells were transfected with a miRNA agomir and a SMAD4 expression vector. The expression of miR125b-5p was determined by using quantitative real-time polymerase chain reaction. Cell apoptosis and necrosis were measured with flow cytometry. The expression of SMAD4 was evaluated with Western blotting. Here, we demonstrated that the rates of apoptosis and necrosis were significantly decreased in the miR125b-5p agomir group of HUVECs under H2O2-induced oxidative stress compared with the miR125b-5p antagomir group. Further experiments revealed that the expression of SMAD4 is negatively regulated by miR125b-5p. Moreover, we identified that the rates of apoptosis and necrosis were increased when SMAD4 and miR125b-5p were both overexpressed compared with miR125b-5p overexpression alone. The present study demonstrates for the first time that the overexpression of miR125b-5p can reduce H2O2-induced oxidative damage via SMAD4, suggesting that miR125b-5p has therapeutic potential for preventing oxidative stress-related diseases.

The pyrrolidinoindoline alkaloid Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling.

AIM: Psm2, one of the pyrrolidinoindoline alkaloids isolated from whole Selaginella moellendorffii plants, has shown a potent antiplatelet activity. In this study, we further evaluated the antiplatelet effects of Psm2, and elucidated the underlying mechanisms. METHODS: Human platelet aggregation in vitro and rat platelet aggregation ex vivo were investigated. Agonist-induced platelet aggregation was measured using a light transmission aggregometer. The antithrombotic effects of Psm2 were evaluated in arteriovenous shunt thrombosis model in rats. To elucidate the mechanisms underlying the antiplatelet activity of Psm2, ELISAs, Western blotting and molecular docking were performed. The bleeding risk of Psm2 administration was assessed in a mouse tail cutting model, and the cytotoxicity of Psm2 was measured with MTT assay in EA.hy926 cells. RESULTS: Psm2 dose-dependently inhibited human platelet aggregation induced by ADP, U4619, thrombin and collagen with IC50 values of 0.64, 0.37, 0.35 and 0.87 mg/mL, respectively. Psm2 (1, 3, 10 mg/kg) administered to rats significantly inhibited platelet aggregation ex vivo induced by ADP. Psm2 (1, 3, 10 mg/mL, iv) administered to rats with the A-V shunt dose-dependently decreased the thrombus formation. Psm2 inhibited platelet adhesion to fibrinogen and collagen with IC50 values of 84.5 and 96.5 mg/mL, respectively, but did not affect the binding of fibrinogen to GPIIb/IIIa. Furthermore, Psm2 inhibited AktSer473 phosphorylation, but did not affect MAPK signaling and Src kinase activation. Molecular docking showed that Psm2 bound to phosphatidylinositol 3-kinase ß (PI3Kß) with a binding free energy of -13.265 kcal/mol. In addition, Psm2 did not cause toxicity in EA.hy926 cells and produced only slight bleeding in a mouse tail cutting model. CONCLUSION: Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling. Psm2 may be a lead compound or drug candidate that could be developed for the prevention or treatment of thrombotic diseases.

[Effect of 2,3-butanedione monoxime on calcium paradox-induced heart injury in rats].

OBJECTIVE: To investigate the Effect of 2,3-butanedione monoxime (BDM) on calcium paradox-induced heart injury and its underlying mechanisms. METHODS: Thirty-two adult male SD rats were randomized into 4 groups, namely the control group, BDM treatment control group, calcium paradox group, and BDM treatment group. Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion and the left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP) were monitored. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Lactate dehydrogenase (LDH) content in the coronary flow was determined. Triphenyltetrazolium chloride staining was used to measure the infarct size, and myocardial cell apoptosis was tested with TUNEL method. Western blotting was used to determine the expression of cleaved caspase-3 and cytochrome c. RESULTS: Compared with the control group, BDM at 20 mmol/L had no effect on cardiac performance, cell death, apoptotic index or the content of LDH, cleaved caspase-3 and cytochrome c at the end of perfusion under control conditions (P>0.05). Calcium paradox treatment significantly decreased the cardiac function and the level of LVDP and induced a larger infarct size (P<0.01), an increased myocardial apoptosis index (P<0.01), and up-regulated expressions of cleaved caspase-3 and cytochrome c (P<0.01). BDM (20 mmol/L) significantly attenuated these effects induced by calcium paradox, and markedly down-regulated the levels of LVEDP and LDH (P<0.01), lowered myocardial apoptosis index, decreased the content of cleaved caspase-3 and cytochrome c (P<0.01), increased LVDP, and reduced the infarct size (P<0.01). CONCLUSION: BDM suppresses cell apoptosis and contracture and improves heart function and cell survival in rat hearts exposed to calcium paradox, suggesting the value of BDM as an potential drug for myocardial ischemia reperfusion injur.

Neolignans from Selaginella moellendorffii.

Two new neolignans selaginellol (1) and selaginellol 4'-O-ß-D-glucopyranoside (2), together with seven known compounds (3-9), were isolated from the whole plant of Selaginella moellendorffii. The structures of the new isolates were determined through spectroscopic data analysis. Compounds 1-9, as well as compounds 10-18 previously isolated from the species, were measured for the activity against platelet aggregation induced by ADP or collagen. Three neoligans (8, 11, and 12), one flavanone (14), and one alkaloid (16) showed inhibitory activity against ADP- or collagen-induced platelet aggregation as compared with tirofiban. The dihydrobenzofuran neolignans (8, 11, and 12) are more potent than the benzofuran neolignan (13) and other types of neolignans (1-7). Glucosidation of the dihydrobenzofuran neolignans (11 and 12) is helpful for the activity. Two new neolignans selaginellol (1) and selaginellol 4'-O-ß-D-glucopyranoside (2) were isolated from the whole plant of Selaginella moellendorffii. Several compounds from this plant showed the activity against platelet aggregation induced by ADP or collagen.

Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling.

Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride-induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kß with the binding free energy of -24.341 kcal/mol, which is close to that of cocrystallized ligand (-24.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases.

Role of KCa3.1 channels in proliferation and migration of vascular smooth muscle cells by diabetic rat serum.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are important events in the development of diabetic atherosclerosis. Previous studies have suggested that K(Ca)3.1 channels participate in atherosclerosis and coronary artery restenosis. In the present study, we attempted to clarify the roles of K(Ca)3.1 channels in the proliferation and migration of VSMCs using experimental type-2 diabetes rat serum and aortic smooth muscle cells (SMC) prepared from non-diabetic rats. mRNA and protein levels and current density of K(Ca)3.1 channels were greatly enhanced in cultured VSMCs treated with diabetic serum. In addition, diabetic serum promoted cell proliferation and migration in cultured VSMCs, and the effects were fully reversed in the cells treated with the K(Ca)3.1 channels blocker TRAM-34. In conclusion, serum from diabetic rats increases the expression of K(Ca)3.1 channels and promotes proliferation and migration of VSMCs to possibly participate in vascular remodeling in diabetes.

KCa3.1 channels mediate the increase of cell migration and proliferation by advanced glycation endproducts in cultured rat vascular smooth muscle cells.

The mechanisms underlying the involvement of advanced glycation endproducts (AGEs) in diabetic atherosclerosis are not fully understood. The present study was designed to investigate whether intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1 channels) are involved in migration and proliferation induced by AGEs in cultured rat vascular smooth muscle cells (VSMCs) using approaches of whole-cell patch voltage-clamp, cell proliferation and migration assay, and western blot analysis. It was found that the current density and protein level of K(Ca)3.1 channels were enhanced in cells incubated with AGE-BSA (bovine serum albumin), and the effects were reversed by co-incubation of AGE-BSA with anti-RAGE (anti-receptors of AGEs) antibody. The ERK1/2 inhibitors PD98059 and U0126, the P38-MAPK inhibitors SB203580 and SB202190, or the PI3K inhibitors LY294002 and wortmannin countered the K(Ca)3.1 channel expression by AGE-BSA. In addition, AGE-BAS increased cell migration and proliferation, and the effects were fully reversed with anti-RAGE antibody, the K(Ca)3.1 channel blocker TRAM-34, or K(Ca)3.1 small interfering RNA. These results demonstrate for the first time that AGEs-induced increase of migration and proliferation is related to the upregulation of K(Ca)3.1 channels in rat VMSCs, and the intracellular signals ERK1/2, P38-MAPK and PI3K are involved in the regulation of K(Ca)3.1 channel expression.

Insulin-mediated upregulation of K(Ca)3.1 channels promotes cell migration and proliferation in rat vascular smooth muscle.

The detailed molecular mechanisms underlying pathogenesis of various vascular diseases such as atherosclerosis are not fully understood in type-2 diabetes. The present study was designed to investigate whether insulin regulates K(Ca)3.1 channels and participates in vasculopathy in type-2 diabetes. A rat model with experimental insulin-resistant type-2 diabetes was used for detecting pathological changes in the aorta wall, and cultured vascular smooth muscle cells (VSMCs) were employed to investigate the regulation of K(Ca)3.1 channels by insulin and roles of K(Ca)3.1 channels in cell migration and proliferation using molecular biology and electrophysiology. Early pathological changes were observed and expression of K(Ca)3.1 channels increased in the aorta wall of the type 2 diabetic rats. K(Ca)3.1 channel mRNA, protein levels and current density were greatly enhanced in cultured VSMCs treated with insulin, and the effects were countered in the cells treated with the ERK1/2 inhibitor PD98059, but not the p38-MAPK inhibitor SB203580. In addition, insulin stimulated cell migration and proliferation in cultured VSMCs, and the effects were fully reversed in the cells treated with the K(Ca)3.1 blocker TRAM-34 or PD98059, but not SB203580. These results demonstrate the novel information that insulin increases expression of K(Ca)3.1 channels by stimulating ERK1/2 phosphorylation thereby promoting migration and proliferation of VSMCs, which likely play at least a partial role in the development of vasculopathy in type-2 diabetes.

Experimental diabetes mellitus down-regulates large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle and alters functional conductance.

Cerebral vascular dysfunction and associated vascular complications often develop over time in type-2 diabetes, but the underlying mechanisms are not wholly understood. The aim of the present study was to investigate whether large-conductance Ca(2+)-activated K(+) (BKCa) channels in cerebral artery smooth muscle cells (CASMCs) were impaired in experimental model of type-2 diabetes, and the changes could account for cerebral vascular complication in type-2 diabetes. Sprague-Dawley rats were fed with high fat and glucose diet for 8 weeks and then injected with streptozotocin (STZ/30 mg/kg i.p.). Three months after injection of STZ, the alterations of BKCa channels were assessed by using multi-myograph system, patch-clamp, RT-PCR and Western blot. Our results show that the model is characterized by insulin resistance, hyperglycaemia, hyperlipidemia and moderate hypertension, which resembles the clinical manifestation of patients with typre-2 diabetes. Inhibition of BKCa channels with 1 mM tetraethylammonium (TEA) or 1 microM paxilline (PAX) causes smaller constriction in type-2 diabetic cerebral basilar arteries than control arteries. The contractile efficacy of 5-Hydroxytryptamine (5-HT) is substantially reduced by TEA or PAX pretreatment in control > diabetic basilar artery rings. The response to 5-HT in diabetic basilar artery rings is higher than that of control artery rings after activation of BKCa channels with NS1619. The whole-cell K(+) currents are significantly decreased in type-2 diabetic CASMCs compared to control, and the sensitivity of BKCa channels to voltage, the specific inhibitor and opener are all diminished in diabetic CASMCs. The expression of BKCa channel beta1, but not alpha-subunits is markedly reduced at both of mRNA and protein levels in endothelial-denudated cerebral arteries. In conclusion, type-2 diabetes downregulates BKCa channel beta1-subunits in CASMCs, resulting in reduced activity of BKCa channel, increased vascular tone and blood pressure, thereby contributing to cerebral vascular complication in type-2 diabetes.

[Identification of up-regulated genes induced by angiotensin II in cardiac fibroblasts].

To identify up-regulated genes in adult rat cardiac fibroblasts (CF) induced by angiotensin II (Ang II), suppression subtractive hybridization (SSH) was performed between the CF stimulated by Ang II (tester) and unstimulated CF (driver) to generate subtractive cDNA library. The library was screened with dot blots hybridization to further verify the differentially expressed cDNA clones. Partial positive clones (19 up-regulated genes) were sequenced and BLAST analyzed. Twelve up-regulated genes related to extracellular matrix, cell cycle, intracellular signal transduction, cell cytoskeleton, cell metabolism and 7 new expressed sequence tags (EST) were acquired (GenBank accession number: CN382808, CN382809, CN382810, CN382811, CN382812, CN382813, CN382814). Our data reveal that SSH is a powerful technique of high sensitivity for the detection and cloning of up-regulated genes expressed in CF induced by Ang II, which may be helpful to clarify the mechanism of cardiac remodeling.