Endothelium-specific overexpression of human IC53 downregulates endothelial nitric oxide synthase activity and elevates systolic blood pressure in mice.

AIMS: Hypertension is one of the major risk factors for cardiovascular diseases. Endothelial cells (ECs) exert important functions in the regulation of blood pressure. A novel gene, IC53, as an isoform of the cyclin-dependent kinase (CDK)-binding protein gene C53, is mainly expressed in vascular ECs and is upregulated in the failing heart of rats. Overexpression of IC53 promotes proliferation of ECs. To examine whether IC53 plays a role in the regulation of vascular tone and blood pressure, we constructed a transgenic (tg) mouse model of the IC53 gene and studied its phenotypes relevant to vascular function. METHODS AND RESULTS: IC53 cDNA was cloned from a human aorta cDNA library. Using the endothelium-specific VE-cadherin promoter, we constructed tg mice in which IC53 was specifically overexpressed in vascular endothelia and found that the tg mice exhibit elevated systolic blood pressure (SBP) in contrast to the wild-type (wt) controls. Further studies revealed impaired endothelium-dependent vasodilation, reduced nitric oxide (NO) production and decreased endothelial NO synthase (eNOS) expression, and activity in the tg mice. Inhibition of IC53 in human umbilical vein ECs induces upregulation of eNOS activity. CONCLUSION: Our results indicate that IC53 participates in the regulation of vascular homeostasis. Endothelium-specific overexpression of IC53 is associated with elevated SBP, which may be in part attributed to the downregulation of eNOS signalling.

[Comparison of distributions of muscle injection and intravenous administration of human mesenchymal stem cells in denervated muscles of the sciatic nerve injured rats].

OBJECTIVE: To explore the better transplantation way of mesenchymal stem cells (MSCs) in neuromuscular disease and its effect on the recovery of injured sciatic nerves. METHODS: Human MSCs were isolated, cultured and fluorescently labeled in vitro. Fifty-four rats were randomly divided into MSC intramuscular (ims), intravenous (iv) and saline administration groups. The sciatic nerve was crashed by hemostat for 5 min. MSCs (10(6)) were injected 3 days after injury. Sciatic nerve function index (SFI), electromyography and muscle biopsy specimens were recorded on ad3, ad7,ad14, ad21, ad28 and ad60. RESULTS: Denervation and regeneration of nerves were observed in the three groups. Spontaneous activity and action potential happened earlier in ims injected group than in the other two groups. The nerve conductivity velocity of ims injected rats was significantly faster than those of the iv group and the control group on d14 [(32.27+/-7.42) m/s,(22.92+/-7.34) m/s and (17.67+/-5.52) m/s; F=5.661,P=0.042]. On d60, the conduction velocity was about the same among the three groups(P>0.05). However, compound muscle action potential (CMAP) amplitude in ims injected group was significantly higher as compared with the other two groups[ims injected group (12.50+/-2.06) mV, iv group (1.50+/-0.20) mV, control group (10.13+/-4.04) mV, F=6.347, P=0.033]. The MSCs were able to be observed only in ims injected tissues 3 weeks after implantation (A large number of small undifferentiated cells were found outside the myofibers and some were found between the cells.) The atrophy of gastrocnemius in ims injected group was much less severe than that of the other 2 groups. The diameter of muscle fibers was significantly longer on d60 (F=4.537, P=0.021). CONCLUSION: Intramuscular injection of MSC was well distributed in denervated muscle, which provides a new way of nerve regeneration in the rat model of sciatic nerve injury.

Gene transfer of heat-shock protein 20 protects against ischemia/reperfusion injury in rat hearts.

AIM: To explore whether overexpression of HSP20 in the myocardium could protect against ischemia/reperfusion injury in rats. METHODS: Rat hearts were injected with vector, recombinant adenovirus encoding green fluorescent protein (Ad.GFP) or recombinant adenovirus encoding wild-type HSP20 (Ad.HSP20) in the left ventricle. Four days later, hearts were removed and expression of HSP20 was measured in the left ventricle. Subsets of animals in the vector-, Ad.GFP- , and Ad.HSP20-treated groups were subjected to 20-min ischemia and 120-min reperfusion. Myocardial injury was evaluated by infarct size and level of serum cardiac troponin T and creatine phosphokinase. Apoptosis of cardiomyocytes was determined by TUNEL staining. Cardiac function was evaluated by hemodynamic indexes. RESULTS: Infarct size and serum cardiac troponin T and creatine phosphokinase levels were significantly reduced in Ad.HSP20-treated hearts compared with vector- and Ad.GFP-treated hearts. The ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes in the Ad.HSP20 group was significantly reduced as compared with the vector and Ad.GFP groups. Left ventricular end systolic pressure, and maximal rate of pressure increase (+dp/dt(max)) and decrease (-dp/dt(min)) values were increased significantly, while left ventricular end diastolic pressure was decreased significantly in Ad.HSP20-treated hearts compared with vector- and Ad.GFP-treated hearts. CONCLUSION: These data indicate that the cardioprotective effects of HSP20 may contribute to the reduction of myocardial necrosis and apoptosis in ischemia/reperfusion injury in rats.

Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute: characteristics and effects on rats.

Conjugate of bovine hemoglobin (bHb) and human serum albumin (HSA) was prepared. The product was simply composed of 89.7% one-to-one Hb-HSA conjugate, 6.0% oligomer of Hb and HSA, 3.5% unconjugated HSA and 0.8% unconjugated Hb, with an average molecular weight of 157 kD. The physicochemical characteristics were determined. Effects of single replacement on blood pressure and long-term survival of rats with 30% and 60% acute blood loss were studied, in comparison with Ringer-lactate solution, stroma-free hemoglobin (SFHb), 5% HSA in Ringer-lactate, whole blood and no resuscitation fluid. Results showed that Hb-HSA conjugate maintained the mean arterial pressure of rats to initial level with no pressor effect. Long-term effects of the replacement fluids on 30% bleeding rats showed that, for the group infused with Hb-HSA conjugate, histology of five major organs, heart, kidney, liver, spleen and lung, were essentially normal, similar to that of whole blood, while obviously renal side-effects appeared in other groups. The efficacy of the conjugate was further demonstrated by the resuscitation of lethal hemorrhagic shock rats (60% acute blood loss) with 100% survival rate (followed for 14 days), the same result as whole blood. The Hb-HSA conjugate can thus be another candidate for blood substitute in emergency.

Adrenomedullin(27-52) inhibits vascular calcification in rats.

Adrenomedullin (ADM) has the vasodilatory properties and involves in the pathogenesis of vascular calcification. ADM could be degraded into more than six fragments in the body, including ADM(27-52), and we suppose the degrading fragments from ADM do the same bioactivities as derived peptides from pro-adrenomedullin. The present study carries forward by assessing the effects on vascular calcification of the systemic administration of ADM(27-52). The rat vascular calcific model was replicated with vitamin D3 and nicotine. ADM or/and ADM(27-52) were systemically administrated with mini-osmotic pump beginning at seventh day after the model replication for 25 days. Vascular calcific nodules histomorphometry, vascular calcium content, vascular calcium uptake, alkaline phosphatase activity, and osteopontin-mRNA quantification in aorta were assessed. ADM limited 40.2% vascular calcific nodules (P<0.01), did not effect on calcium content (P>0.05), reduced 44.4% calcium uptake (P<0.01), lowered 21.1% alkaline phosphatase activity (P<0.01), and regulated 40.9% downwards osteopontin-mRNA expression (P<0.01) in the aorta of rats with vascular calcification. ADM(27-52) receded 32.0% vascular calcific nodules (P<0.01), taken from 55.5% calcium content (P<0.01), did not affect calcium uptake (P>0.05), inhibited 22.5% alkaline phosphatase activity (P<0.01), and restrained 21.9% osteopontin-mRNA expression (P<0.01) in the aorta of rats with vascular calcification. Both of ADM and ADM(27-52) did interact on vascular calcification each other. ADM could partially antagonize the effects of ADM(27-52) in taking from calcium content (17.5%, P<0.01) and in receding vascular calcific nodules (18.6%, P<0.01). ADM could obviously enhance the action of ADM(27-52) in inhibiting alkaline phosphatase activity (14.4%, P<0.01) and in reducing calcium uptake (11.4%, P<0.01). ADM(27-52) could partially antagonize the effects of ADM on regulating downwards osteopontin-mRNA expression (17.0%, P<0.01). It is concluded that ADM(27-52) derived from ADM acts as an inhibitory agent on vascular calcification, with special mechanisms different from ADM derived from ADM progenitor molecule.

Changes of aortae and pulmonary arteries under simulated microgravity and effects of NOS inhibitor on cardiovascular deconditioning.

The present study was to determine the effects of simulated microgravity (SM) on the pulmonary artery (PA) and aorta(TA), and to disclose the changes in pathophysiology of cardiovascular deconditioning(CVD) induced by SM and to explore the effects of NOS inhibitor (N-nitro-L-arginine methylester, L-NAME) on CVD. The high hemodynamics in pulmonary and systemic circulation of human bodies appeared during the initial period and super-regulatory phenomena under 6°head-down tilt bed.

Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression.

Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury. Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion. Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins, Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion, enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group. Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion, expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.

[Changes of reactivity of rabbit femoral venous rings after 21 d simulated weightlessness].

OBJECTIVE: To study the effects of simulated weightlessness on reactivity of leg veins. METHOD: Vascular bathing technique was used to measure the contractile responses of rabbit femoral venous rings to Ht, PE and KCl, and the dilatory responses to ACh and SNP after horizontal confinement or head-down tilt confinement for 21 d. RESULT: After simulated weightlessness for 21 d, the contractile response of rabbit femoral venous rings to higher concentrations of PE and KCl decreased significantly (P<0.05); the contractile response to Ht also decreased, but not significantly (P>0.05); the dilatory responses to ACh and SNP did not change significantly in both groups. CONCLUSION: Contractile response of rabbit femoral vein decreased after simulated weightlessness for 21 d, which might partly explain the occurrence of orthostatic intolerance in men after space flight.

[Impact of simulated microgravity on the expression and distribution of cardiac gap junction protein CX43].

OBJECTIVE: To observe the expression and distribution changes of connexin 43 (CX) in rats' myocardium after simulated microgravity and explore the partial mechanism of arrhythmia. METHOD: Male Wistar rats were randomly assigned to either tail-suspension group (SUS) or control group (CON). Immunohistochemistry and Western Blot were used to detect the expression and the distribution of CX43. Electromicroscope was used to observe the ultrastructural changes. RESULT: In SUS group, the decrease of CX43 and the distribution disturbance were obvious (P<0.05), the proportion of the side-to-side gap junction increased (P<0.05), and space between some gap junctions disappeared. CONCLUSION: The results show that CX43 decreased significantly and distributed irregularly after simulated microgravity. These can cause the changes of cardiac electric conduction velocity and direction. As a result, conduction block and reentry may occur. And these might be the partial mechanism of cardiac arrhythmia.

[The effects of simulated microgravity on pulmonary arteries and aortae].

AIM: Through studying local regulatory mechanisms in pulmonary arteries (PA) and thoracic aortae (TA) under simulated microgravity (SM), to collect some data for the researches of adaptive mechanisms in pulmonary and systemic arteries and for the mechanisms accounting for orthostatic intolerance after SM. METHODS: Cardiopulmonary circulatory function during 7-day 6 degrees head down bed rest (HDT) in male young volunteers was measured with a XXH-2000 pulmonary circulation and cardiac function instrument. - 30 degrees C tail suspended (TS) rats were used as the model to simulate the physiological effects of M. The PA and TA changes of vasoreactivity were respectively observed by vitro vessel rings perfusion. RESULTS: The changes in volume of PA and pulmonary vein during a cardiac cycle and the preload in left cardiac ventricle in men increased significantly in the initial HDT. The super-regulatory phenomena appeared in both pulmonary and systemic circulation, but earlier and more obviously in pulmonary circulation than systemic circulation during 96-144 h. The dilatory reactivity in TS7 PA increased significantly, tended to decrease in TS14. The dilatory reactivity of TA in TS7 had a significant increase, had a slight increase in TS14. The contractile reactivity of PA decreased slightly in TS7 from CON, and were attenuated significantly in TS14. The contractile reactivity of TA in TS14 decreased significantly. The responsiveness to KCl, phenylephrine and sodium nitroprusside in VEC- removed PA had no differences among all groups. CONCLUSION: The differences in changes between pulmonary and systemic arteries under SM could be an important sign of depressed local regulatory function, which might be mainly due to dilatory function in VEC and contribute to the occurrence of orthostatic intolerance after SM.