A Selective TRPC3 Inhibitor Pyr3 Attenuates Myocardial Ischemia/Reperfusion Injury in Mice.

An emerging body of evidence indicates that transient receptor potential TRP channels act as important mediators for a wide variety of physiological functions and are potential targets for drug discovery. Our previous study has identified transient receptor potential channel 3 (TRPC3) and TRPC6 as cation channels through which most of the damaging calcium enters, aggravates pathological changes in vivo and increases ischemia/reperfusion (I/R) injury in mice. This study aimed to verify the effects of TRPC3 inhibitor Pyr3 on myocardial I/R injury in mice. C57BL/6J wild-type male mice (8 to 12 weeks old) were anesthetized with 3.3% chloral hydrate. A murine I (30 min)/R (24 h) injury model was established by temporary occlusion of the left anterior descending (LAD) coronary artery. Pyr3 was administered at concentrations of 0, 2.5, 5, or 10 mg/kg via the right jugular vein 5 min before reperfusion. We observed that the selective TRPC3 inhibitor, 10 mg/kg Pyr3, significantly decreased the infarct size of left ventricle, and reduced the myocardial cell apoptosis rate and inflammatory response in mice. In a conclusion, TRPC3 can function as a candidate target for I/R injury prevention, and Pyr3 may directly bind to TRPC3 channel protein, inhibit TRPC3 channel activity, and improve TRPC3-related myocardial I/R injury. Pyr3 may be used for clarification of TRPC3 functions and for treatments of TRPC3-mediated diseases.

Biomechanical properties of ascending aorta and pulmonary trunk in pigs and humans.

BACKGROUND: This study aims to obtain the biomechanical properties of ascending aorta and pulmonary trunk between healthy humans and pigs of different months, so as to provide necessary biomechanical experimental basis for anastomosing blood vessel in pig-to-human heart xenotransplantation. METHODS: Ascending aorta and pulmonary trunks of the six deceased donors (male 4, female 2) and 42 Chinese Hubei white pigs aged 1-7 months were performed biomechanical test. The blood vessel was given periodic permanent loading and unloading, and repeated force-deformation data were obtained. The elastic properties of the blood vessels were obtained by curve from experimental data. RESULTS: The biomechanical material constant of ascending aorta and pulmonary trunk of pigs did not increase with the increase of age (F = 14.569, P = 0.126). The biomechanical material constant of humans was basically similar to that of pigs aged 1-7 months (F = 12.264, P = 0.225). The elastic modulus was the biggest in pigs aged 7 months in comparison with that in other ages (F = 27.425, P = 0.032). There was no significant difference of elastic modulus of corresponding blood vessel between humans and pigs of different months (F = 17.328, P = 0.215). CONCLUSIONS: Our present study suggests that there was no significant difference of elastic properties of ascending aorta and pulmonary trunks between humans and pigs. From biomechanical aspects, anastomosis of corresponding ascending aorta and pulmonary trunks in the process of pig-to-human heart xenotransplantation may be feasible.

Quantitative analysis of the microstructure of human umbilical vein for assessing feasibility as vessel substitute.

We explored the feasibility of human umbilical vein (HUV) as a small-caliber vessel substitute. HUVs of 50 fetuses were collected on spontaneous miscarriage or labor with the pregnant women's permission. Gestational age ranged 24-42 weeks, and parturients were 20-30 years old. Each sample was sliced into 5 mum frozen transverse sections and stained with hematoxylin-eosin (HE), Weigert, aniline blue, and orange yellow G. The geometric morphological indexes and microstructural component were measured by a computer image analysis system. The media thickness was 0.186, 0.203, 0.237, 0.264, and 0.268 mm at 24-27, 28-32, 33-36, 37-40, and 41-42 weeks, respectively (F = 133.35, p < 0.01); diameters were 1.861, 1.962, 2.303, 2.464, and 2.465 mm (F = 37.35, p < 0.01), respectively. The media thickness and diameter of HUVs increased with gestational age. The elastin content of media increased at 24-40 weeks, but the collagen content and collagen/elastin (C/E) ratio decreased. Elastin content in the proximal segment was higher than in the distal segment [10.16, 6.36 Aa%, (Aa% is the unit of relative content, ie, the ratio of absolute areas to the total tested area of smooth muscle, collagen and elastin in the vascular wall) F = 5.77-12.3, p < 0.05], with the collagen to elastin (C/E) ratio increasing from the proximal to the distal segment (F = 7.63-13.4, p < 0.05). Our results suggest that the microstructural component of HUVs (2.0-3.0 mm caliber) at 37-40 weeks of gestation was similar to that of the small-caliber arteries and had moderate amounts of collagen and elastin and good elasticity, i.e., a good C/E ratio; therefore, HUV may be a substitute for small-caliber arteries (e.g., brachial, ulnar, radial, right coronary, anterior tibial, and posterior tibial). HUV is one of several graft materials that may be used when autogenous saphenous vein is absent or inadequate.

Coculture with endothelial cells enhances vascular smooth muscle cell adhesion and spreading via activation of beta1-integrin and phosphatidylinositol 3-kinase/Akt.

The interactions between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) play significant roles in the homeostasis of the blood vessel during vascular remodeling. Cell adhesion and spreading are an essential process for VSMC migration, survival and proliferation in the events of vascular physiology and pathophysiology. However, effects of ECs on adhesion and spreading of VSMCs have not been characterized yet. Here, the interaction of ECs and VSMCs on adhesion and spreading of VSMCs were investigated by using a coculture system. The results showed that VSMCs cocultured with ECs exhibited a significant increase in the number of adherent and spreading cells, and much more mRNA (twofold, P<0.01) and protein (threefold, P<0.05) expression of beta(1)-integrin comparing to the control, i.e., VSMCs cultured alone. Furthermore, the enhanced functional activity of beta(1)-integrin expression was confirmed by FACS. A beta(1)-integrin blocking antibody (P5D2) could inhibit the EC-induced VSMC adhesion and spreading. It was demonstrated that in correspondence with enhanced cell adhesion, ECs also prompted focal adhesion complex assembly and stress fiber formation of VSMCs. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway was more pronouncedly activated in response to VSMC attachment. Our results for the first time show that coculture with ECs enhances VSMC adhesion and spreading by up-regulating beta(1)-integrin expression and activating the PI3K/Akt pathway, suggesting that the interaction between ECs and VSMCs serves an important role in vascular homeostasis and remodeling.

Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain.

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway.

Shear stress protects against endothelial regulation of vascular smooth muscle cell migration in a coculture system.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress; these forces strongly influence the behaviors of neighboring vascular smooth muscle cells (VSMCs). VSMC migration is a key event in vascular wall remodeling. In this study, the authors assessed the difference between VSMC migration in VSMC/EC coculture under static and shear stress conditions. Utilizing a parallel-plate coculture flow chamber system and Transwell migration assays, they demonstrated that human ECs cocultured with VSMCs under static conditions induced VSMC migration, whereas laminar shear stress (1.5 Pa, 15 dynes/cm2) applied to the EC side for 12 h significantly inhibited this process. The changes in VSMC migration is mainly dependent on the close interactions between ECs and VSMCs. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-mediated EC regulation of VSMC migration. Wortmannin and Akti significantly inhibited the EC-induced effect on VSMC Akt phosphorylation and migration. These results indicate that shear stress protects against endothelial regulation of VSMC migration, which may be an atheroprotective function on the vessel wall.

miR-155 functions downstream of angiotensin II receptor subtype 1 and calcineurin to regulate cardiac hypertrophy.

Cardiac hypertrophy is characterized by maladaptive tissue remodeling that may lead to heart failure or sudden death. MicroRNAs (miRs) are negative regulators of angiotensin II and the angiotensin II receptor subtype 1 (AGTR1), which are two components involved in cardiac hypertrophy. In the present study, the interaction between angiotensin II receptor subtype 1 (AGTR1) signaling and miR-155 was investigated. Rat H9C2 (2-1) cardiomyocytes were transfected with miR-155 analogues or inhibitors, then stimulated with angiotensin II to induce cardiac hypertrophy. miR-155 expression was revealed to be altered following transfection with chemically-modified miR-155 analogues and inhibitors in rat cardiomyocytes. In cell cardiac hypertrophy models, the cell surface area, AGTR1, atrial natriuretic peptide and myosin heavy chain-ß mRNA expression levels were revealed to be lower in cells stimulated with miR-155 analogue-transfected cells treated with angiotensin II compared with cells stimulated with angiotensin alone (P<0.05), as determined using reverse transcription-polymerase chain reaction (PCR), quantitative PCR and western blot analyses. Furthermore, calcineurin mRNA and protein, intracellular free calcium and nuclear factor of activated T-cells-4 proteins were downregulated in miR-155 analogue-transfected cells treated with angiotensin II, as compared with cells stimulated with angiotensin II alone (P<0.05). In conclusion, the current study indicates that miR-155 may improve cardiac hypertrophy by downregulating AGTR1 and suppressing the calcium signaling pathways activated by AGTR1.

[Effects of adenoviral vector containing human angiotensin II type 1 receptor antisense cDNA on biological action of human pulmonary artery smooth muscle cells].

OBJECTIVE: To investigate the effect of human angiotensin II (AngII) type 1 receptor (AT(1)R) antisense cDNA (ahAT(1)) on migration, proliferation, and apoptosis of cultured human pulmonary artery smooth muscle cells (PASMC). METHODS: Two recombinant adenoviral vectors, AdCMVahAT(1) containing full length antisense cDNA targeting to human AT(1)R mRNA, and AdCMVLacZ containing LacZ, were constructed by orientation clone technology and homologous recombination. The PASMC was divided into 3 groups (DMEM, AdCMVLacZ, AdCMVahAT(1)) and different interventions were given to different groups. AT(1)R expression was detected by RT-PCR and immunohistochemistry method; migration of PASMC was measured by Boyden's Chamer method. Other PASMC was divided into 4 groups (DMEM, AngII, AdCMVLacZ + AngII and AdCMVahAT(1) + AngII), and only the last 2 groups were respectively transfected with AdCMVLacZ and AdCMVahAT(1) before administration of AngII. From 6 h to 96 h after stimulation by AngII (10(-7) mol/L), proliferation index (PI) and apoptosis of PASMC were determined by flow cytometry. RESULTS: At the 48 h the level of AT(1)R mRNA was significantly less in PASMC transfected AdCMVahAT(1) than that in group DMEM and in group AdCMVLacZ. The protein level showed a same difference (P < 0.01). At 24 h the migration distance of PASMC also was significantly less in group AdCMVahAT(1) than that in group DMEM and Group AdCMVLacZ (P < 0.01). Stimulated by AngII for 48 h, in group AngII the PI of PASMC markedly increased (P < 0.01 vs group DMEM). But in Group AdCMVahAT(1) + AngII PI of PASMC clearly decreased (P < 0.01 vs group AngII and group DMEM respectively). There was no statistic difference of PI between group AdCMVLacZ + AngII and group AngII. Moreover, apoptosis peak emerged only in group AdCMVahAT(1) + AngII. The rate of apoptosis in those PASMC used AdCMVahAT(1) and AngII was 24.70 +/- 4.04 (P < 0.01 vs the other 3 groups respectively). CONCLUSIONS: These results indicate that AngII stimulates proliferation via AT(1) receptors in human PASMC, and antisense cDNA targeting to human AT(1)R transfection mediated by adenoviral vector has powerful inhibitory effects on AngII-induced migration and proliferation of human PASMC by attenuating AT(1)R mRNA and protein expression. Also, it can promote apoptosis of human PASMC. That demonstrate that AT(1)R antisense cDNA is a potent inhibitors of the actions of AngII on PASMC. Antisense inhibition targeting to AT(1)R has therapeutic potential for the treatment of pulmonary vascular diseases.

Effects of dihydrotestosterone on adhesion and proliferation via PI3-K/Akt signaling in endothelial progenitor cells.

The protective effects of male hormones on the cardiovascular system are still in dispute. There is now ample evidence that testosterone level is negatively correlated to the incidence and mortality of cardiovascular disease in men. Endothelial progenitor cells (EPCs) play a vital role in endothelial healing and vascular integrity, which are useful for promoting cardiovascular health. In this study, we investigated the effects of dihydrotestosterone (DHT), a non-aromatizable androgen, on human EPC function and the activation of the phosphatidylinositol-3-kinase (PI3-K)/Akt pathway in vitro. EPCs were incubated with a series of concentrations (1, 10, or 100 nmol/L in DMSO) of DHT for 24 h or with 10 nmol/L DHT for different time (6, 12, 24, 48 h). EPC adhesion and proliferation and the activation of Akt were assayed by cell counting, 5-ethynyl-2'-deoxyuridine incorporation assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blot analysis. Our data demonstrated that DHT significantly increased the proliferative activity and adhesive ability of EPCs in a dose- and time-dependent manner, maximum at 10 nmol/L, 24 h (p < 0.05). Western blot analysis revealed that DHT promoted the phosphorylation of Akt, and the effects of different concentrations of DHT on Akt phosphorylation were consistent with those on EPC proliferation and adhesion (p < 0.05). However, the enhancing effects of DHT on EPCs decreased with administration of the pharmacological PI3-K blocker LY294002 (p < 0.05). In conclusion, DHT can modulate EPC proliferation and adhesion and the PI3-K/Akt pathway plays an important role in this process.

Effect of sex on biomechanical properties of the proper hepatic artery in pigs and humans for liver xenotransplant.

OBJECTIVES: This study investigated the relation between biomechanical properties of the proper hepatic artery and sex in pigs and humans to provide the theoretical basis for selecting suitable donor in pig-to-human liver xenotransplant. MATERIALS AND METHODS: The proper hepatic arteries of 32 Chinese Hubei white pigs (8 males, 8 females, 8 castrated males, and 8 ovariectomized females) and 10 deceased donors (5 human men, 5 human women) were obtained. The pressure-diameter relations of the proper hepatic arteries were measured on biomechanical test equipment to calculate the incremental elastic modulus (Einc), pressure-strain elastic modulus (Ep), volume elastic modulus (Ev), and compliance. Each sample was sliced into 5-µm frozen sections and stained with hematoxylin-eosin. RESULTS: There were significant differences in Einc (F=10.24; P = .001), Ep (F=3.75; P = .001), and Ev (F=3.41; P = .002) of the proper hepatic arteries of female, male, and gonadectomized pigs; females had the lowest elastic modulus and the gonadectomized group had the highest (P < .01). There was a significant difference in compliance of the porcine proper hepatic arteries between the sexes, highest in the female group and lowest in the gonadectomized group (P < .01). No difference in the elastic modulus and compliance of the proper hepatic artery between the male pig and the human man. There was no difference between the female pig and the human woman. CONCLUSIONS: There were differences in the biomechanical properties of the proper hepatic arteries of the female, male, and gonadectomized pigs. The biomechanical properties of the human men/women proper hepatic artery match those of the porcine male/female hepatic artery. The correlation between sex and biomechanical properties of the proper hepatic artery in pigs could imply that a pig of the same sex should be chosen for pig-to-human liver xenotransplant.

Morphological and biomechanical remodelling of the hepatic artery in a swine model of portal hypertension.

OBJECTIVES: To obtain the biomechanical and morphological remodelling of hepatic arteries in swine with portal hypertension. METHODS: A number of 20 white pigs was used, of which 14 were subjected to liver cirrhosis and portal hypertension (PHT) induced by carbon tetrachloride and pentobarbital; the rest were used as the control group. The biomechanical remodelling of the hepatic arteries was measured, namely, the incremental elastic modulus (E inc), pressure-strain elastic modulus (E p), volume elastic modulus (E v), the incremental compliance (C), the opening angle and the stained microstructural components of the vessels. RESULTS: The percentages for the microstructural components and the histologic data significantly changed in the experimental group, three incremental elastic moduli (E inc, E p, and E v) of the experimental group were significantly larger than those of the control group (P < 0.05); the compliance of hepatic arteries decreased greatly (P < 0.05) too. The opening angle (OA) was considerably larger than that of control group (P < 0.05). CONCLUSIONS: The study suggests that the morphological and biomechanical properties of swine hepatic arteries have changed significantly during the process of portal hypertension and that from biomechanical aspects, the hepatic arteries have also suffered from extensive remodelling, which in turn deteriorates the existing portal hypertension.

Pim-1 is up-regulated by shear stress and is involved in shear stress-induced proliferation of rat mesenchymal stem cells.

AIMS: Investigation of the response of mesenchymal stem cells (MSCs) to vascular mechanical forces is very important in the field of cardiovascular intervention. Ser/Thr-protein kinase Pim-1 is a novel transducer of cell survival and the cell cycle that promotes signals in the hematopoietic cell system. Current studies aim to foster an understanding of Pim-1 expression and regulation in MSCs in response to different durations and strengths of laminar shear stress (SS) and to investigate the role of Pim-1 in SS-induced cell proliferation. MAIN METHODS: A parallel-plate flow chamber was used to control the strength and duration of SS. Proliferation was measured with the BrdU cell proliferation assay. The expressions of Pim-1 mRNA and protein were evaluated by reverse transcription-polymerase chain reaction and western blotting, respectively. RNA interference was used to knock down the Pim-1 gene. KEY FINDINGS: The results showed that SS up-regulation of Pim-1 mRNA and protein was time-dependent. Pim-1 induction was SS strength-dependent, and the expression level reached a maximum at 30 dynes/cm(2). Inhibitors of p38MAPK and ERK attenuated the SS-induced expression of Pim-1. In addition, SS significantly increased BrdU-uptake, which was effectively blocked by the silencing of Pim-1. SIGNIFICANCE: These results demonstrated that Pim-1 is expressed in MSCs and plays an important role in the SS-induced proliferation of MSCs.

Involvement of Akt1/protein kinase Balpha in tumor conditioned medium-induced endothelial cell migration and survival in vitro.

PURPOSE: Endothelial cell migration and survival might be called "major angiogenic responses". Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in angiogenesis models in vitro. However, the molecular events triggered by tumor CM are not fully understood. Here, we examined the effects of the CM from human lung carcinoma cell lines A549 and SPC-A-1 on cultures of primary human umbilical veins endothelial cells (HUVECs). METHODS: After treatment of HUVECs with the CM, cell migration was assessed by wound-healing assay, cell viability was evaluated by XTT assay, and apoptosis and cell death of HUVECs was analyzed by flow cytometry. Phosphorylation of Akt was assessed by Western blotting. To dissect the direct role of Akt, small interfering RNA (siRNA) against Akt1 was used. RESULTS: Both A549 and SPC-A-1 CM significantly stimulated cell migration. However, only A549 CM promoted cell viability and inhibited low serum-induced apoptosis and cell death of HUVECs, but SPC-A-1-CM showed no effects on survival of HUVECs. Meanwhile, A549 CM was found to be able to induce much more phosphorylation of Akt compared to SPC-A-1 CM treated group. The inhibitor of PI3K (wortmaninn) or Akt1 siRNA blocked A549 CM-induced migration and survival of HUVECs. CONCLUSION: These results indicated that the angiogenic effects of A549 CM are largely mediated through activation of the PI3K-Akt in endothelial cells, and that the Akt1 is crucial in this process, which may provide a therapeutic target for decreasing tumor angiogenesis.