Berberine hydrochloride inhibits migration ability via increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Inadequate trophoblasts migration and invasion is considered as an initial event resulting in preeclampsia, which is closely related to oxidative stress. Berberine hydrochloride (BBR), extracted from the traditional medicinal plant Coptis chinensis Franch., exerts a diversity of pharmacological effects, and the crude drug has been widely taken by most Chinese women to treat nausea and vomit during pregnancy. But there is no research regarding its effects on trophoblast cell function. AIM OF THE STUDY: This study aimed to investigate the effect of BBR on human-trophoblast-derived cell line (HTR-8/SVneo) migration ability and its mechanism. MATERIALS AND METHODS: Cell viability was detected by CCK-8 assay. The effect of BBR on cells migration function was examined by scratch wound healing assay and transwell migration assay. Intracellular nitric oxide (NO), superoxide (O2-) and peroxynitrite (ONOO-) levels were measured by flow cytometry. The expression levels of inducible NO synthase (iNOS), eNOS, p-eNOS, MnSOD, CuZnSOD, Rac1, NOX1, TLR4, nuclear factor-κB (NF-κB), p-NFκB, pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) in cells were analyzed by Western blotting. Uric acid sodium salt (UA), the scavenger of ONOO-, PEG-SOD (a specific superoxide scavenger), L-NAME (a NOS inhibitor) and antioxidants (Vit E and DFO) were further used to characterize the pathway of BBR action. RESULTS: 5 µM BBR decreased both the migration distance and the number of migrated cells without affecting cells viability in HTR-8/SVneo cells after 24 h treatment. BBR could increase the level of NO in HTR-8/SVneo cells, and the over-production of NO might be attributable to iNOS, but not eNOS. BBR could increase intracellular O2- levels, and the over-production of O2- is closely related with Rac1 in HTR-8/SVneo cells. The excessive production of NO and O2- further react to form ONOO-, and the increased ONOO- level induced by BBR was blunted by UA. Moreover, UA improved the impaired migration function caused by BBR in HTR-8/SVneo cells. The depressed migration function stimulated by BBR in HTR-8/SVneo cells was diminished by PEG-SOD and L-NAME. Furthermore, BBR increased the expression of IL-6 in HTR-8/SVneo cells, and antioxidants (Vit E and DFO) could decrease the expression of IL-6 and iNOS induced by BBR. CONCLUSIONS: BBR inhibits the cell migration ability through increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells, indicating that BBR and traditional Chinese medicines containing a high proportion of BBR should be used with caution in pregnant women.

Preventive Cold Acclimation Augments the Reparative Function of Endothelial Progenitor Cells in Mice.

BACKGROUND/AIMS: Chronic cold exposure may increase energy expenditure and contribute to counteracting obesity, an important risk factor for cerebrocardiovascular diseases. This study sought to evaluate whether preventive cold acclimation before ischemia onset might be a promising option for preventing cerebral ischemic injury. METHODS: After a 14-day cold acclimation period, young and aged mice were subjected to permanent cerebral ischemia, and histological analyses and behavioral tests were performed. Mouse endothelial progenitor cells (EPCs) were isolated, their function and number were determined, and the effects of EPC transplantation on cerebral ischemic injury were investigated. RESULTS: Preventive cold acclimation before ischemia onset increased EPC function, promoted ischemic brain angiogenesis, protected against cerebral ischemic injury, and improved long-term stroke outcomes in young mice. In addition, transplanted EPCs from cold-exposed mice had a greater ability to reduce cerebral ischemic injury and promote local angiogenesis compared to those from control mice, and EPCs from donor animals could integrate into the recipient ischemic murine brain. Furthermore, transplanted EPCs might exert paracrine effects on cerebral ischemic injury, which could be improved by preventive cold acclimation. Moreover, preventive cold acclimation could also enhance EPC function, promote local angiogenesis, and protect against cerebral ischemic injury in aged mice. CONCLUSIONS: Preventive cold acclimation before ischemia onset improved long-term stroke outcomes in mice at least in part via promoting the reparative function of EPC. Our findings imply that a variable indoor environment with frequent cold exposure might benefit individuals at high risk for stroke.

GTP cyclohydrolase I prevents diabetic-impaired endothelial progenitor cells and wound healing by suppressing oxidative stress/thrombospondin-1.

Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level.

Hydrogen sulfide improves wound healing via restoration of endothelial progenitor cell functions and activation of angiopoietin-1 in type 2 diabetes.

Impaired angiogenesis and its induced refractory wound lesions are common complications of diabetes. Hydrogen sulfide (H2S) has been reported to have proangiogenic effects. We hypothesize that H2S improves diabetic wound healing by restoring endothelial progenitor cell (EPC) function in type 2 diabetes. db/db Mice were treated with sodium hydrosulfide (NaHS), 4-hydro-xythiobenzamide group (HTB), or saline for 18 days. db/+ Mice were treated with dl-propargylglycine (PAG) or saline for 18 days. Plasma H2S levels were significantly decreased in db/db mice and restored in the NaHS and HTB mice compared with the diabetic control group. Wound-closure rates were significantly faster in the NaHS and HTB groups than in the db/db group, in which the PAG group had slower wound-closure rates. Wound skin capillary densities were enhanced in the NaHS and HTB groups. EPC functions were significantly preserved in the NaHS and HTB groups but were decreased in the PAG group. Meanwhile, EPC functions of the db/+ mice were significantly reduced after in vitro PAG treatment or cystathionine-γ-lyase (CSE) silencing; EPC functions of db/db mice were significantly improved after in vitro NaHS treatment. The expressions of Ang-1 in wound skin tissue and in EPCs were upregulated in the NaHS and HTB groups compared with db/db controls, but were downregulated by in vivo PAG and in vitro siCSE treatment compared with normal controls. Diabetic EPC tube formation capacity was significantly inhibited by Ang-1 small interfering RNA before NaHS treatment compared with db/db EPCs treated with NaHS only. Taken together, these results show that H2S improves wound healing by restoration of EPC functions and activation of Ang-1 in type 2 diabetic mice.

A potential role of alpha-7 nicotinic acetylcholine receptor in cardiac angiogenesis in a pressure-overload rat model.

This work investigated the expression of alpha-7 nicotinic acetylcholine receptor (α7nAChR) in the left ventricle and its putative role in cardiac angiogenesis in a pressure overload rat model induced by abdominal aorta coarctation. Blood pressure and protein levels of α7nAChR were measured at 4, 8, 12, and 16 weeks after surgery. mRNA levels of α7nAChR, cardiac vagus nerve function, cardiac hypertrophy, and microvessel density of the left ventricle were determined at the final 16-week period. The role of α7nAChR in angiogenesis was evaluated. It was found that systolic blood pressure above the coarctation site was greater at 16 weeks after coarctation and expression of α7nAChR was significantly increased at both mRNA and protein levels in the left ventricle compared with the control. Positive staining for receptors was mainly focused around vessels and among the degenerated cardiomyocytes. Cardiac vagus nerve function was significantly attenuated; microvessel density was markedly increased and was associated with cardiac hypertrophy. Activation of α7nAChR induced tube formation of cultured human umbilical vein endothelial cells (HUVECs). We conclude that expression of α7nAChR was increased at 16 weeks after coarctation, and this might be a compensatory response to decreased vagus nerve function and cardiac hypertrophy and may also play a role in cardiac angiogenesis.

GTP cyclohydrolase I/BH4 pathway protects EPCs via suppressing oxidative stress and thrombospondin-1 in salt-sensitive hypertension.

Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O2⁻) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS(-/-)/GCH(+/-) hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O2⁻ in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O2⁻ levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.

Blood pressure variability, cardiac baroreflex sensitivity and organ damage in experimentally hypertensive rats.

1. The present study was designed to investigate the haemodynamic features and morphological changes in experimentally hypertensive rat models. 2. Sprague-Dawley rats were used to prepare the experimentally hypertensive models, including two-kidney, one-clip renovascular hypertensive (2K1C) rats, deoxycorticosterone acetate salt hypertensive (DOCA) rats and N(G)-nitro-l-arginine methyl ester-induced hypertensive (l-NAME) rats. Six weeks after the induction of hypertension, 24 h blood pressure was recorded and blood pressure variability (BPV) expressed by 24 h (or 12 h in the daytime and night-time study) standard deviation of the variables was calculated. Then, cardiac baroreflex sensitivity (BRS) was determined and four endogenous factors (tumour necrosis factor-alpha, interleukin-1beta, angiotensin II and endothelin-1) were measured. Finally, morphological changes were examined. 3. It was found that an increase in BPV and a decrease in BRS were accompanied by an elevation of blood pressure in all three hypertensive models. The DOCA rats had the highest BPV, whereas the l-NAME rats had the lowest BRS. 4. Morphological changes were similar in DOCA and l-NAME rats and the cardiac changes were relatively slight in 2K1C rats. Tumour necrosis factor-alpha was increased in all the three models, especially in DOCA rats. Endothelin-1 was higher in DOCA rats and angiotensin II was increased in 2K1C rats and decreased in DOCA rats. 5. In conclusion, increased BPV and decreased BRS accompanied the elevation of blood pressure in all three hypertensive models. The DOCA rats had the highest BPV and the l-NAME rats had the lowest BRS. Obvious organ damage was seen in all three hypertensive models 6 weeks after the induction of hypertension.

Frequent ventricular premature beats increase blood pressure variability in rats.

AIM: The present study was designed to test a hypothesis that nonfatal ventricular arrhythmia such as ventricular premature beats (VPB) is a contributing factor in the elevation of blood pressure variability (BPV). METHODS: Blood pressure (BP) and electrocardiogram were continuously recorded. The relation between VPB and BPV was observed under conscious state in chronic myocardial infarction (MI) rats one month after ligation of the left coronary artery, and further verified under anesthetized state in rat model of ventricular arrhythmia produced by acute intravenous infusion of aconitine. RESULTS: MI rats exhibited a big difference in the count and pattern of VPB, and were divided into no VPB, occasional VPB, and frequent VPB groups. Among the three groups, there were no differences in BP, heart period (HP), and MI size. However, BPV was markedly higher in frequent not occasional VPB rats, and HP variability (HPV) was larger in both frequent and occasional VPB rats, when compared with no VPB rats. In the whole population of MI rats, BPV was positively correlated with VPB and HPV, not with BP, HP and MI size. Infusion of aconitine had no effect on BP, HP, BPV, and HPV during the period without VPB. Frequent VPB after several minutes of aconitine infusion induced significant increase in BPV and HPV with no change in BP and HP. BPV was also positively correlated with VPB and HPV, not with BP and HP. Hemodynamics in aconitine-evoked ventricular tachycardia was characterized as lower BP, higher BPV, and higher HPV. CONCLUSION: High BPV can be caused by frequent not occasional VPB in rats.

Inhibition of inflammation contributes to organ protection of atenolol in sinoaortic-denervated rats.

The present study was designed to test the hypothesis that inhibition of inflammation contributes to the protective effects of atenolol on the organ damage induced by sinoaortic denervation (SAD) in rats. SAD was performed in male Sprague-Dawley rats at the age of 10 weeks. Atenolol (20 mg/kg/d, po) was administered for 12 weeks beginning from 4 weeks after SAD. Organ damage evaluation and the determination of plasma TXB2, serum IL-1, TNF-alpha and tissue reactive oxygen species (ROS) were performed at 16 weeks after SAD. It was found that there existed obvious organ damage including increased cardiac and aortic collagen, and glomerular injury, in SAD rats. Plasma TXB2, serum TNF-alpha IL-1, and tissue ROS increased significantly after SAD. Long-term treatment with atenolol significantly prevented the organ damage with a decrease in left ventricular weight, cardiac and aortic collagen contents, and glomerular injury score in SAD rats. Plasma TXB2, serum IL-1, and tissue ROS were found to be significantly decreased by the long-term treatment with atenolol. Furthermore, it was found that the levels of inflammation-related factors were significantly related to all the organ-damage parameters studied in this experiment. These results suggest that inhibition of inflammation and oxygen stress contributes to the organ-protective effects of atenolol in SAD rats.

Inflammation is involved in the organ damage induced by sinoaortic denervation in rats.

OBJECTIVE: The present study was designed to test the hypothesis that inflammation is involved in the end-organ damage (EOD) induced by sinoaortic denervation (SAD) in rats. METHOD: SAD was performed in male Sprague-Dawley rats at the age of 10 weeks. Under anaesthesia, aortic nerves were cut and the sinus region of the carotid artery was stripped and painted with 10% phenol. Pathological evaluation of EOD and the determination of plasma or tissue levels of the factors related to inflammation, including thromboxane B2 (TXB2) interleukin-1 (IL-1), tumour necrosis factor alpha (TNF-alpha) and reactive oxygen species (ROS) were performed at 16 weeks after SAD. Pathological evaluation of EOD included heart weight ratio, myocardial and blood vessel hydroxyproline and collagen volume fraction, glomerular injury score and number of infiltrating inflammatory cells. Indomethacin (20 mg/kg per day, orally) or vitamin E (100 mg/kg per day, orally) was administered for 12 weeks, beginning from 4 weeks after SAD, to observe their effects on SAD-induced EOD. RESULTS: There were significant fibrosis and inflammatory infiltration in the myocardium and blood vessels, represented by higher hydroxyproline and collagen volume fraction, and a large amount of inflammatory cells in the tissues of SAD rats. Heart weight and kidney glomerular injury score were significantly higher in SAD than in sham-operated rats. Plasma TXB2, TNF-alpha, IL-1 and tissue ROS increased significantly after SAD. Indomethacin and vitamin E significantly decreased the contents of some factors related to inflammation in SAD rats. Both drugs also alleviated myocardial and vessel fibrosis, inflammatory infiltration and kidney damage. CONCLUSION: Inflammation is involved in the organ damage induced by SAD in rats.

Candesartan inhibits sinoaortic denervation-induced cardiovascular hypertrophy in rats.

AIM: To study the effect of candesartan cilexetil (candesartan), a new AT1 receptor antagonist, on sinoaortic denervation (SAD)-induced cardiovascular hypertrophy and its potential mechanisms in rats. METHODS: For long-term treatment, candesartan (6 mg/kg/d) was given in rat food for 16 weeks after SAD surgery, and for acute treatment, a single dose of candesartan (3 mg/kg) was administrated intragastrically at 30 d after SAD. RESULTS: The indexes of left ventricular and aortic hypertrophy in candesartan-treated SAD rats were decreased when compared with untreated SAD rats, and similar to or less than those in normal rats. SAD-induced cardiomyocyte hypertrophy, myocardial fibrosis, wall thickening of intramyocardial arterioles and aortae, and destruction of vascular internal elastin membrane were almost inhibited by candesartan. The plasma angiotensin II levels were markedly increased in treated SAD rats and negatively correlated with the indexes of hypertrophy. Both blood pressure and its variability were reduced by a single dose of candesartan during 3 h of observation period. CONCLUSION: Candesartan can efficiently inhibit SAD-induced cardiovascular hypertrophy. In addition to known mechanisms, upregulation of circulating angiotensin II and stabilization of blood pressure may be involved in this cardiovascular protection of candesartan.