Low-dose nifedipine rescues impaired endothelial progenitor cell-mediated angiogenesis in diabetic mice.

It is of great clinical significance to develop potential novel strategies to prevent diabetic cardiovascular complications. Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic vascular complications. In the present study we evaluated whether low-dose nifedipine could rescue impaired EPC-mediated angiogenesis and prevent cardiovascular complications in diabetic mice. Diabetes was induced in mice by five consecutive injections of streptozotocin (STZ, 60 mg·kg-1·d-1, i.p.). Diabetic mice were treated with low-dose nifedipine (1.5 mg·kg-1·d-1, i.g.) for six weeks. Then, circulating EPCs in the peripheral blood were quantified, and bone marrow-derived EPCs (BM-EPCs) were prepared. We showed that administration of low-dose nifedipine significantly increased circulating EPCs, improved BM-EPCs function, promoted angiogenesis, and reduced the cerebral ischemic injury in diabetic mice. Furthermore, we found that low-dose nifedipine significantly increased endothelial nitric oxide synthase (eNOS) expression and intracellular NO levels, and decreased the levels of intracellular O2.- and thrombospondin-1/2 (TSP-1/2, a potent angiogenesis inhibitor) in BM-EPCs of diabetic mice. In cultured BM-EPCs, co-treatment with nifedipine (0.1, 1 µM) dose-dependently protected against high-glucose-induced impairment of migration, and suppressed high-glucose-induced TSP-1 secretion and superoxide overproduction. In mice with middle cerebral artery occlusion, intravenous injection of diabetic BM-EPCs treated with nifedipine displayed a greater ability to promote local angiogenesis and reduce cerebral ischemic injury compared to injection of diabetic BM-EPCs treated with vehicle, and the donor-derived BM-EPCs homed to the recipient ischemic brain. In conclusion, low-dose nifedipine can enhance EPCs' angiogenic potential and protect against cerebral ischemic injury in diabetic mice. It is implied that chronic treatment with low-dose nifedipine may be a safe and economic manner to prevent ischemic diseases (including stroke) in diabetes.

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.

A preventive injection of endothelial progenitor cells prolongs lifespan in stroke-prone spontaneously hypertensive rats.

There is a pressing need for new approaches to prevent stroke. Endothelial progenitor cells (EPCs) promote vascular repair and revascularization in the ischemic brain. The present study sought to evaluate whether preventive delivery of EPCs could prevent or protect against stroke. Stroke-prone spontaneously hypertensive rats (SHR-SP) received a single injection of EPCs, and their survival time was monitored. In addition, at 28 and/or 42 days after a single injection of EPCs, SHR-SP and mice were subjected to cerebral ischemia, and cerebral ischemic injury, local angiogenesis and in vivo EPC integration were determined. Other experiments examined the effects of EPC conditioned medium, and the distribution of donor EPCs taken from GFP transgenic mice. It was found that EPC-pretreated SHR-SP showed longer lifespans than untreated controls. A single preventive injection of EPCs could produce persistent protective effects against cerebral ischemic injury (lasting at least 42 days), and promote local angiogenesis in the ischemic brain, in two types of animals (SHR-SP and normotensive mice). EPCs of donor origin could be detected in the recipient peripheral blood, and integrated into the recipient ischemic brains. Furthermore, it was suggested that mouse EPCs might exert paracrine effects on cerebral ischemic injury in addition to their direct angiogenic effects. In conclusion, a single preventive injection of EPCs prolonged the lifespan of SHR-SP, and protected against cerebral ischemic injury for at least 7 weeks. It is implied that EPC injection might be a promising candidate for a preventive role in patients at high risk for stroke.

Combination Therapy of Nifedipine and Sulphonylureas Exhibits a Mutual Antagonistic Effect on the Endothelial Cell Dysfunction Induced by Hyperglycemia Linked to Vascular Disease.

BACKGROUND/AIMS: By inducing severe endothelial impairment, hypertension and diabetes are two leading causes of morbidity and mortality. Hypertensive patients with concomitant diabetes must take both antihypertensive and hypoglycaemic medications, for which there is a lack of experimental and clinical guidelines. This study aimed to examine the interaction between these two types of medication on the endothelial cell function. METHODS: The effect of antihypertensive (nifedipine and irbesartan) and anti-diabetic (metformin and glibenclamide/glimepiride) drugs on human umbilical vein cells (HUVECs) function was examined using a modified Boyden chamber assay. The intracellular NO and O2- levels of HUVECs were detected through flow cytometry. RESULTS: Our findings showed that nifedipine/sulphonylurea monotherapy significantly attenuated high glucose-induced (33 mM) HUVECs migration incapacity, while combination therapy of nifedipine and glibenclamide/glimepiride showed no protective effect. Both nifedipine/metformin monotherapy and combined therapy significantly mitigated the migration incapacity induced by high glucose in HUVECs. Combined with either metformin or sulphonylureas, irbesartan therapy was able to attenuate the high glucose-induced migration incapacity of HUVECs. Nifedipine monotherapy decreased the O2- levels and increased the NO levels in in vitro-cultured HUVECs treated with high glucose. However, the combination therapy of nifedipine and glibenclamide increased the O2- levels and decreased the NO levels compared to the nifedipine monotherapeutic group. CONCLUSION: The nifedipine and glibenclamide/glimepiride combination exerted a mutual antagonistic effect on the protection from high glucose-induced impairment in endothelial cells, which might be partially attributed to the increased O2- level and decreased NO level. These results imply that calcium channel blockers + sulphonylurea combination therapy warrants further attention in patients suffering from both hypertension and diabetes.

Prolonged Fasting Improves Endothelial Progenitor Cell-Mediated Ischemic Angiogenesis in Mice.

BACKGROUND/AIMS: Prolonged fasting (PF) was shown to be of great potency to promote optimal health and reduce the risk of many chronic diseases. This study sought to determine the effect of PF on the endothelial progenitor cell (EPC)-mediated angiogenesis in the ischemic brain and cerebral ischemic injury in mice. METHODS: Mice were subjected to PF or periodic PF after cerebral ischemia, and histological analysis and behavioral tests were performed. Mouse EPCs were isolated and examined, and the effects of EPC transplantation on cerebral ischemic injury were investigated in mice. RESULTS: It was found that PF significantly increased the EPC functions and angiogenesis in the ischemic brain, and attenuated the cerebral ischemic injury in mice that was previously subjected to cerebral ischemia. Periodic PF might reduce cortical atrophy and improve long-term neurobehavioral outcomes after cerebral ischemia in mice. The eNOS and MnSOD expression and intracellular NO level were increased, and TSP-2 expression and intracellular O2- level were reduced in EPCs from PF-treated mice compared to control. In addition, transplanted EPCs might home into ischemic brain, and the EPCs from PF-treated mice had a stronger ability to promote angiogenesis in ischemic brain and reduce cerebral ischemic injury compared to the EPCs from control mice. The EPC-conditioned media from PF-treated mice exerted a stronger effect on cerebral ischemic injury reduction compared to that from control mice. CONCLUSION: Prolonged fasting promoted EPC-mediated ischemic angiogenesis and improved long-term stroke outcomes in mice. It is implied that prolonged fasting might potentially be an option to treat ischemic vascular diseases.

Dietary intake of sugar substitutes aggravates cerebral ischemic injury and impairs endothelial progenitor cells in mice.

BACKGROUND AND PURPOSE: In our current food supply, sugar substitutes are widely used in beverages and other food products. However, there is limited information about the link between dietary consumption of sugar substitutes and stroke to date. This study sought to determine the effect of various sugar substitutes on the cerebral ischemic injury and endothelial progenitor cells, which have been implicated to play an important role in vascular repair and revascularization in ischemic brain tissues, in mice. METHODS: After treatment with sucrose and various sugar substitutes (the doses are in the range of corresponding acceptable daily intake levels) and vehicle for 6 weeks, mice were subjected to permanent left middle cerebral artery occlusion, and the infarct volumes, angiogenesis, and neurobehavioral outcomes were determined. In addition, the number and function of endothelial progenitor cells were also examined. RESULTS: After long-term treatment with fructose, erythritol (sugar alcohols), acesulfame K (artificial sweeteners), or rebaudioside A (rare sugars), the cerebral ischemic injury (both infarct volumes and neurobehavioral outcomes) was significantly aggravated, angiogenesis in ischemic brain was reduced, and endothelial progenitor cell function was impaired in mice compared with control. However, the similar impairments were not found in sucrose (with the same dose as fructose's)-treated mice. CONCLUSIONS: Long-term consumption of sugar substitutes aggravated cerebral ischemic injury in mice, which might be partly attributed to the impairment of endothelial progenitor cells and the reduction of angiogenesis in ischemic brain. This result implies that dietary intake of sugar substitutes warrants further attention in daily life.

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.

Sodium dichloroacetate improves migration ability by suppressing LPS-induced inflammation in HTR-8/SVneo cells via the TLR4/NF-κB pathway.

Objectives: Inadequate cytotrophoblast migration and invasion are speculated to result in preeclampsia, which is a pro-inflammatory condition. Sodium dichloroacetate (DCA) exerts anti-inflammatory actions. Thus,we sought to investigate the effect of DCA on the migration function of the lipopolysaccharide (LPS)-stimulated human-trophoblast-derived cell line (HTR-8/SVneo). Materials and Methods: HTR-8/SVneo cells were treated with LPS to suppress cell migration. Cell migration was examined by both scratch wound healing assay and transwell migration assay. Western blotting was used to analyze the expression levels of toll-like receptor-4 (TLR4), nuclear factor-κB (NF-κB), TNF-α, IL-1ß, and IL-6 in the cells. Results: DCA reversed LPS-induced inhibition of migration in HTR-8/SVneo cells. Furthermore, DCA significantly suppressed LPS-induced activation of TLR4, phosphorylation of NF-κB (p65), translocation of p65 into the nucleus, and the production of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). Treatment with inhibitors of TLR4 signal transduction (CLI095 or MD2-TLR-4-IN-1) reduced LPS-induced overexpression of pro-inflammatory cytokines, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells. Conclusion: DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least in part, via the TLR4/NF-κB signaling pathway. This result indicates that DCA might be a potential therapeutic candidate for human pregnancy-related complications associated with trophoblast disorder.

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.

Synergism of irbesartan and amlodipine on hemodynamic amelioration and organ protection in spontaneously hypertensive rats.

AIM: To investigate the synergism of low-doses of amlodipine and irbesartan on reduction of blood pressure variability (BPV), amelioration of baroreflex sensitivity (BRS) and organ protection in spontaneously hypertensive rats (SHR). METHODS: The rats were administered amlodipine (1 mg·kg(-1)·d(-1)) alone, irbesartan (10 mg·kg(-1)·d(-1)) alone, or the combination of the two drugs for 4 months. The drugs were mixed into the rat chow. Blood pressure (BP) was continuously monitored in conscious animals. After the determination of BRS, the rats were killed for morphological evaluation of organ damages. RESULTS: The combination of low-dose irbesartan and amlodipine had statistically significant synergism on reduction of BP and BPV, amelioration of BRS and organ protection in SHR. Multiple regression analysis showed that the decrease in left ventricular hypertrophy was associated with the decrease in systolic BPV (r=0.665, P<0.01); the decrease in aortic hypertrophy was associated with the increase in BRS (r=0.656, P<0.01); and the amelioration in renal lesion was associated with the increase in BRS (r=0.763, P<0.01) and the decrease in systolic BPV (r=0.706, P<0.01). CONCLUSION: Long-term treatment with a combination of low-doses of amlodipine and irbesartan showed significant synergism on reduction of BP and BPV, restoration of BRS and organ protection in SHR. Besides BP reduction, the enhancement of BRS and reduction of BPV might contribute to the organ protection.

Low-Dose Piperlongumine Rescues Impaired Function of Endothelial Progenitor Cells and Reduces Cerebral Ischemic Injury in High-Fat Diet-Fed Mice.

It is of great clinical significance to develop potential novel strategies to prevent cardio-cerebrovascular complications in patients with hyperlipidemia. Vascular Endothelial integrity and function play a key role in the prevention of cardio-cerebrovascular diseases. Endothelial progenitor cells (EPCs) can home to sites of ischemic injury and promote endothelial regeneration and neovascularization. Hypercholesterolemia impairs the function of EPC. The present study attempted to identify the effect of piperlongumine on EPCs' angiogenic potential and cerebral ischemic injury in high-fat diet-fed (HFD-fed) mice. Here, we showed that treatment with low-does piperlongumine (0.25 mg/kg/day) for 8 weeks significantly improved EPCs function and reduced the cerebral ischemic injury (both infarct volumes and neurobehavioral outcomes) in HFD-fed mice. In addition, low-dose piperlongumine administration increased intracellular NO level and reduced intracellular O2 - level in EPCs of HFD-fed mice. Moreover, incubation with piperlongumine (1.0 µM, 24 h) reduced thrombospondin-1/2 (TSP-1/2, a potent angiogenesis inhibitor) expression levels in EPCs from HFD-fed mice, increased the therapeutic effect of EPC from HFD-fed mice on cerebral ischemic injury reduction and angiogenesis promotion in HFD-fed mice, and the donor derived EPCs homed to the recipient ischemic brain. In conclusion, low-dose piperlongumine can enhance EPCs' angiogenic potential and protect against cerebral ischemic injury in HFD-fed mice. It is implied that treatment with low-dose piperlongumine might be a potential option to prevent ischemic diseases (including stroke) in patients with hyperlipidemia, and priming with piperlongumine might be a feasible way to improve the efficacy of EPC-based therapy for ischemic diseases.

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.

Effects of low-dose ketanserin on atherosclerosis in rats and rabbits.

The present study was designed to test the hypothesis that a small dose of ketanserin, which enhances baroreflex activity, prevents the early lesions of atherosclerosis. In experiment 1, baroreflex sensitivity (BRS) was measured in 31 spontaneously hypertensive rats (SHRs) in a conscious state using a computerized blood pressure monitoring system. Four weeks later, the rats were administered vitamin D3 and fed a high-cholesterol diet for 8 weeks to induce atherosclerosis. Then their hearts and aortae were removed for pathological examination. A negative correlation was found between BRS and the scores of coronary (r = -0.460, P < 0.01) and aortic atherosclerosis (r = -0.448, P < 0.05) in SHR. In experiment 2, SHRs were divided into 3 groups (n = 10 in each group) and received a dose of ketanserin of 0.3, 1.0, and 3.0 mg/kg (i.g.), respectively. At the smallest dose (0.3 mg/kg), ketanserin did not lower blood pressure but enhanced BRS. In experiment 3, SHRs were administered vitamin D3, fed a high-cholesterol diet, and simultaneously treated with low-dose ketanserin. The atherosclerosis scores of the treatment group were significantly lower than those of the control group (coronary score: 0.90 ± 0.14 vs. 1.76 ± 0.27, P < 0.05; aortic scores: 1.00 ± 0.39 vs. 2.18 ± 0.41, P < 0.05). In experiment 4, male New Zealand White rabbits were fed a high-cholesterol diet and treated with low-dose ketanserin at the same time. The atherosclerosis scores of the treatment group were significantly lower than those of the control group (aortic scores: 0.26 ± 0.20 vs. 0.60 ± 0.31, P < 0.05). In conclusion, the present study demonstrated, for the first time, that low-dose ketanserin prevented the development of atherosclerosis independent of its blood pressure lowering action in SHRs and New Zealand White rabbits at least in part via enhancement of arterial baroreflex function.

The combination of atenolol and amlodipine is better than their monotherapy for preventing end-organ damage in different types of hypertension in rats.

Combinations therapy is often used in hypertensive patients whether combination therapy is necessary for preventing end-organ damage is not known. The objective of this study was to determine in four different hypertensive animal models the necessity of adding the calcium channel blocker amlodipine to therapy with the ss-blocker atenolol to modulate end-organ damage. Spontaneously hypertensive rats, DOCA-salt hypertensive rats, two-kidney, one-clip renovascular hypertensive rats and Lyon genetically hypertensive rats were used to study this objective. These animal models have different sensitivities to atenolol and amlodipine. The dosages of therapy employed were 10 mg/kg atenolol alone, 1 mg/kg amlodipine, 10 mg atenolol + 1 mg/kg amlodipine and 5 mg/kg atenolol+0.5 mg/kg amlodipine. BP was continuously recorded in all animals. After determination of baroreflex sensitivity, rats were sacrificed for end-organ damage evaluation. The combination of amlodipine and atenolol had a synergistic inhibitory effect on blood pressure and blood pressure variability, and end-organ damage as compared with monotherapy with atenolol or amlodipine in all animal models. Baroreflex sensitivity also improved with the combination therapy more than with monotherapy. In conclusion, atenolol and amlodipine combination exerts a superior effect on blood pressure, blood pressure variability, baroreflex sensitivity and end-organ damage. The superior effect of the combination was observed in all four models of hypertension.

Synergism of hydrochlorothiazide and nifedipine on blood pressure variability reduction and organ protection in spontaneously hypertensive rats.

This study was designed to investigate the effects of a hydrochlorothiazide-nifedipine combination on blood pressure (BP), blood pressure variability (BPV), baroreflex sensitivity (BRS), and organ protection in spontaneously hypertensive rats (SHR). The doses used were 10 mg/kg/d for both hydrochlorothiazide and nifedipine, and 10+10 mg/kg/d for the combination of these two drugs. Drugs were mixed into rat chow at the aforementioned doses. SHR were treated for 4 months, and then BP was continuously recorded for 24 h. After the determination of BRS, rats were killed for organ-damage evaluation. It was found that long-term treatment with hydrochlorothiazide, nifedipine or both significantly decreased BP and BPV, enhanced BRS and conferred organ protection in SHR. The combination of hydrochlorothiazide and nifedipine had a significant synergistic effect on BPV reduction, BRS enhancement and organ protection in SHR, whereas no obvious synergism on BP reduction was found. Multiple-regression analysis showed that the decrease in left ventricular and aortic hypertrophy was most closely associated with the decrease in systolic BPV and the increase in BRS, and the amelioration of renal lesions was most closely associated with the increase in BRS. In conclusion, long-term treatment with a combination of hydrochlorothiazide and nifedipine yielded a significantly synergistic effect on BPV reduction, BRS restoration and organ protection in SHR. In addition to BP reduction, the decrease in BPV and the enhancement of BRS may have made important contributions to the observed organ protection.

Reduction of blood pressure variability by combination therapy in spontaneously hypertensive rats.

OBJECTIVE: To test the hypothesis that combination therapy might be a powerful tool to reduce blood pressure variability (BPV) in the treatment of hypertension. METHODS: In chronic studies, male spontaneously hypertensive rats were given drugs in their rat chow for 18 weeks (n = 9 or 10 for each group of rats). Doses were as follows: atenolol (10 mg/kg per day), amlodipine (1 mg/kg per day), a combination of atenolol and amlodipine (2.5 + 0.25, 5 + 0.5, and 10 + 1 mg/kg per day), hydrochlorothiazide (8 mg/kg per day), enalapril (3.2 mg/kg per day), and a combination of hydrochlorothiazide and enalapril (2 + 0.8, 4 + 1.6, and 8 + 3.2 mg/kg per day). In acute studies, drugs were perfused through the left femoral vein in conscious rats. RESULTS: In chronic studies, compared with monotherapy, the combinations of two antihypertensive drugs were more effective in reducing the blood pressure (BP), BPV, and organ damage. The indexes of organ damage were all positively related to BP and/or BPV. In acute studies, the constant infusion of phenylephrine (6.25 microg/kg per min) markedly increased the BP, but showed no significant effects on BPV. The infusion of a combination of atenolol and amlodipine (62.5 + 6.25 microg/kg per min) or a combination of hydrochlorothiazide and enalapril (500 + 200 microg/kg per min) significantly reduced the BP and BPV; moreover, a significant reduction in BPV was still found when the rat's BP was restored to control levels by a concomitant infusion of phenylephrine. CONCLUSIONS: Combination therapy may be a powerful and useful tool for BPV reduction in the treatment of hypertension. In addition to the BP reduction, the decrease in BPV may contribute significantly to the prevention of organ damage in hypertension.

Chronic Exposure to Subtherapeutic Antibiotics Aggravates Ischemic Stroke Outcome in Mice.

Subtherapeutic antibiotics have been widely used in agriculture since the 1950s, which can be accumulated in human body through various approaches and may have long-term consequences. However, there is limited information about the link between chronic subtherapeutic antibiotic exposure and the outcome of ischemic brain injury. Here we showed that long-term treatment with subtherapeutic chlortetracycline, penicillin or vancomycin, which were widely used in agriculture approved by US Food and Drug Administration (FDA), could impair EPC functions, reduce ischemic brain angiogenesis and aggravate cerebral ischemic injury and long-term stroke outcomes in mice. In addition, transplantated EPCs from chronic antibiotic-treated mice showed a lower therapeutic effect on cerebral ischemic injury reduction and local angiogenesis promotion compared to those from control mice, and EPCs from the donor animals could integrate into the recipient ischemic brain in mice. Furthermore, transplanted EPCs might exert paracrine effects on cerebral ischemic injury reduction in mice, which could be impaired by chronic antibiotic exposure. In conclusion, chronic subtherapeutic antibiotic exposure aggravated cerebral ischemic injury in mice, which might be partly attributed to the impairment of both EPC-mediated angiogenesis and EPCs' paracrine effects. These findings reveal a previously unrecognized impact of chronic subtherapeutic antibiotic exposure on ischemic injury.

Blood pressure variability is more important than blood pressure level in determination of end-organ damage in rats.

OBJECTIVE: This study was designed to determine how important a novel risk factor of elevated blood pressure variability (BPV) is in the determination of end-organ damage by comparison with the classic risk factor of a high blood pressure (BP) level. METHODS AND RESULTS: The effects of haemodynamics on cardiovascular morphology were evaluated by univariate and multivariate regression analysis in two different rat models with an enlarged distribution of haemodynamics. In male sham-operated and sinoaortic-denervated Wistar-Kyoto rats and spontaneously hypertensive rats (n = 34), BPV was more important than BP in cardiac and renal damage and aortic hypertrophy. BPV and BP had independent effects, explaining 59.4% of the variation in damage to these organs. In male (n = 44) and female (n = 46) F1 hybrids of Sprague-Dawley rats and spontaneously hypertensive rats, the greater importance of BPV than BP was further demonstrated in left ventricular hypertrophy, glomerular damage and aortic hypertrophy. The phenomenon was more evident in females than males for cardiovascular hypertrophy. BPV and BP or BPV alone had independent effects, explaining 46.9% (male) or 37.5% (female) of the variation in damage to these organs. CONCLUSION: BPV is a more critical determinant than BP level for cardiac damage, renal lesions and aortic hypertrophy in rats, strongly suggesting the significance of BPV control for the protection of these organs.

Blood pressure variability, baroreflex sensitivity and organ damage in spontaneously hypertensive rats treated with various antihypertensive drugs.

Besides blood pressure, blood pressure variability and baroreflex sensitivity maybe important factors determining organ damage in hypertension. This study was designed to investigate the effects of various antihypertensive drugs on blood pressure and blood pressure variability reductions, baroreflex sensitivity, and target organ damage in spontaneously hypertensive rats (SHR). The dose is 20 mg/kg/day for atenolol, and 10 mg/kg/day for nifedipine, irbesartan and hydrochlorothiazide. We used relatively low doses of drugs to avoid a very remarkable normalization of blood pressure in the treatment, which would make it much difficult to distinguish the contribution of blood pressure variability and baroreflex sensitivity to organ protection from that of blood pressure. Drugs at the aforementioned doses were mixed into rat chow. SHR were treated for 4 months. Blood pressure was then continuously recorded for 24 h. After the determination of baroreflex sensitivity, rats were killed for organ-damage evaluation. It was found that long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide all markedly reduced blood pressure variability, enhanced baroreflex sensitivity, and produced significant organ protection. Compared with blood pressure level, blood pressure variability and baroreflex sensitivity values showed a much closer or similar relationship with organ-damage parameters in every treatment group of rats. Multiple-regression analysis showed that the decrease in left ventricular hypertrophy, the decrease in aortic hypertrophy and the amelioration in renal lesion were all most closely correlated with the increase in baroreflex sensitivity and the decrease in systolic blood pressure variability. In conclusion, long-term treatment with atenolol, nifedipine, irbesartan or hydrochlorothiazide produced organ protection in SHR. Besides the blood pressure reduction, the decrease in blood pressure variability and the restoration of baroreflex sensitivity may contribute to this organ protection.

Arterial baroreflex dysfunction promotes atherosclerosis in rats.

The present study was designed to test the hypothesis that arterial baroreflex dysfunction promotes the development of atherosclerosis. Experiment 1: the baroreflex sensitivity (BRS) was measured in 30 Sprague-Dawley (SD) rats in conscious state with a computerized blood pressure monitoring system. Four weeks later, the rats were administered with Vitamin D3, and fed with the high-cholesterol diet for 8 weeks to induce atherosclerosis. The hearts and aortae were removed for pathological examination. A negative correlation was found between BRS and the scores of coronary (r=-0.464, P<0.01) or aortic atherosclerosis (r=-0.524, P<0.01) in SD rats. Experiment 2: sinoaortic denervation (SAD) was performed in SD rats. Then atherosclerosis was also induced. The atherosclerosis scores in SAD rats were significantly higher than those in sham-operated rats (aortic score: 1.50+/-0.41 versus 1.10+/-0.39, P<0.05; coronary score: 1.70+/-0.35 versus 1.25+/-0.54, P<0.05). Using immunohistochemistry and Western blotting methods, it was found that the expressions of C-reactive protein, intercellular adhesion molecule-1 and vascular-cell adhesion molecule-1 in coronary artery and aorta were increased in SAD rats compared with sham-operated rats. These results indicate that arterial baroreflex dysfunction promotes the development of atherosclerosis in rats, and that inflammation may be involved in this process.