Zhuyu Annao decoction promotes angiogenesis in mice with cerebral hemorrhage by inhibiting the activity of PHD3.

Some traditional Chinese decoctions, such as Zhuyu Annao, exert favorable therapeutic effects on acute cerebral hemorrhage, hemorrhagic stroke, and other neurological diseases, but the underlying mechanism remains unclear. This study aimed to determine whether Zhuyu Annao decoction (ZYAND) protects the injured brain by promoting angiogenesis following intracerebral hemorrhage (ICH) and elucidate its specific mechanism. The effect of ZYAND on the nervous system of mice after ICH was explored through behavioral experiments, such as the Morris water maze and Rotarod tests, and its effects on oxidative stress were explored by detecting several oxidative stress markers, including malondialdehyde, nitric oxide, glutathione peroxidase, and superoxide dismutase. Real-time quantitative RT-PCR and WB were used to detect the effects of ZYAND on the levels of prolyl hydroxylase domain 3 (PHD3), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) in the brain tissues of mice. The effect of ZYAND on the NF-κB signaling pathway was detected using a luciferase reporter gene. A human umbilical cord vascular endothelial cell angiogenesis experiment was performed to determine whether ZYAND promotes angiogenesis. The Morris water maze test and other behavioral experiments verified that ZYAND improved the neurobehavior of mice after ICH. ZYAND activated the PHD3/HIF-1α signaling pathway, inhibiting the oxidative damage caused by ICH. In angiogenesis experiments, it was found that ZYAND promoted VEGF-induced angiogenesis by upregulating the expression of HIF-1α, and NF-κB signaling regulated the expression of HIF-1α by inhibiting PHD3. ZYAND exerts a reparative effect on brain tissue damaged after ICH through the NF-κB/ PHD3/HIF-1α/VEGF signaling axis.

HIF prolyl hydroxylase inhibitors prevent neuronal death induced by mitochondrial toxins: therapeutic implications for Huntington's disease and Alzheimer's disease.

Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions, but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a "knock-in" mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1alpha mRNA is dramatically induced by mutant huntingtin, HIF-1alpha depletion by short interfering RNAs (siRNA) does not affect steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover, 3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIF-dependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity. Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1alpha gene expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing cell death in conditions such as Huntington's disease and Alzheimer's disease that are associated with metabolic stress in the central nervous system.

HIF prolyl-4-hydroxylase interacting proteins: consequences for drug targeting.

Protein stability of hypoxia-inducible factor (HIF)alpha subunits is regulated by the oxygen-sensing prolyl-4-hydroxylase domain (PHD) enzymes. Under oxygen-limited conditions, HIFalpha subunits are stabilized and form active HIF transcription factors that induce a large number of genes involved in adaptation to hypoxic conditions with physiological implications for erythropoiesis, angiogenesis, cardiovascular function and cellular metabolism. Oxygen-sensing is regulated by the co-substrate-dependent activity and hypoxia-inducible abundance of the PHD enzymes which trigger HIFalpha stability even under low oxygen conditions. Because HIFalpha itself is notoriously reluctant to the development of antagonists, an increase in PHD activity would offer an interesting alternative to the development of drugs that interfere specifically with the HIF signalling pathway. Interestingly, among the recently discovered PHD interacting proteins were not only novel downstream targets but also upstream regulators of PHDs. Their PHD isoform-specific interaction offers the possibility to target distinct PHD isoforms and their non-identical downstream signalling pathways. This review summarizes our current knowledge on PHD interacting proteins, including upstream regulators, chaperonins, scaffolding proteins, and novel downstream transcription factors.

Hypoxia-inducible factor-1 is central to cardioprotection: a new paradigm for ischemic preconditioning.

BACKGROUND: Ischemic preconditioning provides strong cardioprotection from ischemia, but its molecular mechanisms remain unknown. Convincing evidence confirms a central role of hypoxia-inducible factor (HIF)-1 in mammalian oxygen homeostasis. Thus, we pursued HIF-1 as a central component of cardioprotection by ischemic preconditioning. METHODS AND RESULTS: Murine studies of in situ preconditioning revealed a robust activation of cardiac HIF-1. Moreover, in vivo small interfering RNA repression of cardiac HIF-1 resulted in abolished cardioprotection by ischemic preconditioning. In contrast, pretreatment with the HIF activator dimethyloxalylglycine was associated with cardioprotection similar to that of ischemic preconditioning itself. Finally, selective small interfering RNA repression of prolylhydroxylase 2 resulted in significant activation of HIF-1 alpha and attenuated myocardial infarct sizes (0.44+/-0.09-fold). As an end point of HIF-dependent cardioprotection, we defined the role of A2B adenosine receptor (A2BAR) signaling. Although the cardiac A2BAR was induced with HIF activation, HIF-dependent cardioprotection was abolished in A2BAR-/- mice. CONCLUSION: Taken together, these studies provide evidence for a critical role of HIF-1 in ischemic preconditioning via enhancing purinergic signaling pathways.

TNF-alpha suppresses prolyl-4-hydroxylase alpha1 expression via the ASK1-JNK-NonO pathway.

BACKGROUND: Inflammation is known to contribute to the pathogenesis of vascular diseases in which arterial wall extracellular matrix (ECM) homeostasis is disrupted. Tumor necrosis factor-alpha (TNF-alpha), a pivotal cytokine that regulates ECM metabolism by increasing degradation and decreasing production of arterial collagens, is associated with vulnerable plaques and aortic aneurysms. METHODS AND RESULTS: In the current study, we showed that, when administered in doses of 1 to 100 ng/mL, TNF-alpha dose-dependently downregulated the expression of prolyl-4-hydroxylase alphaI [P4H alpha(I)]-the rate-limiting subunit for the P4H enzyme essential for procollagen hydroxylation, secretion, and deposition in primary human aortic smooth muscle cells (HASMCs). Using a progressive deletion cloning approach, we characterized the TNF-alpha-responsive element (TaRE) in the human P4H alpha(I) promoter and found that a negative regulatory region at the position of -32 to +18 bp is responsible for approximately 80% of TNF-alpha-mediated suppression. Using oligonucleotide-based transcription factor pull-down method in which proteins were resolved in 1-D gel electrophoresis and identified using LC-MS/MS, we identified the NonO protein binds this region. When NonO expression silenced with specific siRNA, we found that 70% of the TNF-alpha-mediated P4H alpha suppression was abolished, which appeared to be mediated by the ASK1-JNK pathway. CONCLUSIONS: Our findings define a novel molecular pathway for inflammation associated extracellular matrix dysregulation, which may account for atherosclerotic plaque rupture and aortic aneurysm formation. Further understanding of this pathway may facilitate development of novel therapeutics for vascular diseases.

Inhibition of prolyl 4-hydroxylase prevents left ventricular remodelling in rats with thoracic aortic banding.

BACKGROUND: Pressure overload leads to myocardial remodelling with collagen accumulation, left ventricular hypertrophy (LVH), neurohormonal activation and myocardial dysfunction. Prolyl 4-hydroxylases (P4H) are involved in collagen maturation. Inhibition of P4H has been shown to prevent LV remodelling and improve survival post-myocardial infarction. AIM: To evaluate the role of P4H in pressure overload-induced myocardial remodelling. METHODS: Male Wistar rats underwent thoracic aortic banding (AoB) and were treated with a P4H inhibitor (P4HI) or vehicle (control). Echocardiography and haemodynamic measurements were performed after 4 weeks. Collagens, matrix metalloproteinases (MMP), tissue inhibitors of MMPs (TIMP), growth factors and neurohormonal markers were quantitated in LV samples. RESULTS: AoB led to LVH, increased LV enddiastolic pressure (LVEDP) and decreased contractility compared to sham. P4HI reversed these effects. AoB increased collagen I and III expression, which was normalized by P4HI. AoB led to deregulation of matrix remodelling enzymes, enhanced expression of growth factors and activation of the endothelin system. P4HI partially prevented deregulation of the MMP/TIMP system, inhibited upregulation of growth factors and normalized AoB-induced ECE-1 and ETB expression. CONCLUSIONS: P4HI leads to an improvement of AoB-associated LV dysfunction and reduces imbalance of extracellular matrix turnover and hypertrophy-associated gene expression. P4H inhibition could therefore be of value in treatment of myocardial remodelling accompanying pressure overload hypertrophy.

Transforming growth factor-beta-induced transition of fibroblasts: a model for myofibroblast procurement in tissue valve engineering.

BACKGROUND AND AIM OF THE STUDY: The selection of a suitable cell type for scaffold seeding, its isolation and adequate expansion in vitro remains a major challenge in tissue valve engineering. The study aim was to establish a model for efficient procurement of myofibroblasts for in-vitro seeding using fibroblasts as progenitor cells. METHODS: Dermal and arterial mesenchymal cells from human (hDMC1.1 and hAMC1.1) and sheep (sDMC1.1 and sAMC1.1) were isolated by sequential culture. Cell isolates were characterized by stringent criteria based on morphology, immunocytochemistry using antibodies to vimentin, cytokeratin, prolyl 4-hydroxylase, smooth muscle alpha-actin (alpha-SMA) and smooth muscle myosin, and by Western blotting for alpha-SMA and N-cadherin. Cultures with less than 10-20% alpha-SMA-positive cells were considered to be fibroblastic. Cells were subsequently transdifferentiated with the cytokine transforming growth factor-beta1 (TGF-beta1) during five days, and then evaluated morphotypically, by immunocytochemistry, and by Western blotting. The metabolic and functional properties of TGF-beta1-treated and untreated colonies were compared by measuring the expression of extracellular proteins (collagen type 1 and tenascin-C) and by a collagen matrix contraction assay. RESULTS: TGF-beta1 successfully transformed both human and sheep fibroblasts to metabolically active and functional myofibroblasts based on stringent criteria for myofibroblast characterization. Alpha-SMA positivity of 100% was obtained in all cases (hDMC1.1, hAMC1.1, sDMC1.1, and sAMC1.1) after transformation compared to less than 50% in the non-transformed state (hAMC1.1, 17%; hDMC1.1, 10%; sAMC1.1, 43%; and sDMC1.1, 30%). This observation was further supported by increased contractility and an up-regulation of extracellular protein production in transdifferentiated cells. CONCLUSION: Untreated arterial cell isolates were, at best, less than 50% alpha-SMA-positive. By allowing procurement of high densities of myofibroblasts in a relatively short time, the model was seen to be a potentially useful tool in tissue valve engineering, at least in investigations using autologous cells in the sheep model.

Wound healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin, with special reference to prolylhydroxylase and collagenase enzyme activity.

The healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin were investigated with reference to the enzyme activity of both prolylhydroxylase and collagenase as related to histological findings. The rats were observed by endoscopy on the 3rd day after the subserosal injection of acetic acid; rats with ulcers were divided into three groups: non-treated, and cimetidine- and calcitonin-treated. The latter two groups were treated for 7 days. Prolylhydroxylase activity in active ulcers in the non-treated group was slightly higher on the 3rd day and significantly higher on the 10th day than the activity in control rats that had received subserosal injections of physiological saline solution on the respective days. In non-treated rats, the healed ulcer on the 10th day showed lower prolylhydroxylase activity than that in the active ulcer on the same day. Cimetidine did not affect prolylhydroxylase activity, but, with calcitonin, there was higher prolylhydroxylase activity in the healed than in the active ulcer, although the difference was not significant. Interstitial collagenase showed the highest activity on the 3rd day and decreased on the 10th day in non-treated rats. Collagenase activity was higher in the cimetidine-treated group, than that in the non-treated group, and numerous peroxidase-positive granulocytes were seen in the mucosa and submucosa. Calcitonin did not affect collagenase activity. The participation of both enzymes is indispensable in the healing process and the effects of anti-ulcer agents on these enzymes must be considered.

Tranilast, a selective inhibitor of collagen synthesis in human skin fibroblasts.

Effects of tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 microM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor beta 1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of pro alpha 1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF beta 1-mediated increase in pro alpha 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGF beta 1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug.

Osteolathyrogenic effects of malathion in Xenopus embryos.

Malathion, an organophosphorus insecticide, has been found previously to cause developmental defects such as enlargement of the atria and aorta and bent notochord in Xenopus laevis. Since these defects are similar to those caused by known lathyrogens, the effects of malathion on collagen biochemistry and structure were studied. Embyros were exposed to malathion or its metabolite malaoxon during the first 4 days of development. Notochords of malathion- and malaoxon-treated embryos were bent ventrally between the third and sixth somites and were enlarged. Ultrastructural examination of the postanal tail notochord showed that the elastic externa was disorganized and less dense and the sheath had fewer, more disorganized fibers. Embryos exposed in culture displayed a concentration-dependent reduction in ascorbate and hydroxyproline. Malathion and malaoxon inhibited the activities of lysyl oxidase (I50s of 0.7 and 8.7 nM, respectively) and proline hydroxylase (I50s of 58 microM and 49.9 nM, respectively) in homogenates of Xenopus embryos. These data suggest that malathion and malaoxon alter posttranslational modification of collagen, with resultant morphological defects in connective tissue.

Effects of niacin on bleomycin-induced increases in myeloperoxidase, prolyl hydroxylase, and superoxide dismutase activities and collagen accumulation in the lungs of hamsters.

It has been shown that lung nicotinamide adenine dinucleotide (NAD) depletion accompanies bleomycin (BL)-induced lung fibrosis in the hamster and that treatment with niacin (NA), a precursor of NAD, was found to attenuate lung fibrosis caused by this agent. Niacin was used in the present study to investigate changes in some biochemical parameters and enzymes involved in the development of BL-induced lung fibrosis in the hamster. Niacin (500 mg/kg, IP), or an equivalent volume of saline (SA, IP), was given daily 2 days prior to intratracheal instillation of BL (7.5 U/5 mL/kg) or SA and everyday thereafter throughout the study. Hamsters were killed at 1, 4, 7, 10, and 14 days after the BL or SA instillation and their lungs processed for various biochemical assays. Hydroxyproline content and superoxide dismutase (SOD) activity in SABL treated animals were significantly (P < or = 0.05) elevated at 7 and 10 days, peaking at 14 days to 161 +/- 11% and 159 +/- 11% of the SASA treated animals, respectively. Although the hydroxyproline level of NABL treated animals was significantly elevated at 7 and 10 days and peaked at 14 days to 123 +/- 8% of the NASA control, these values were significantly lower than the SABL treated animals at the corresponding times. The lung SOD activity of NABL groups was significantly higher at 4 days but significantly lower at 10 and 14 days than the SABL groups at the corresponding times. Prolyl hydroxylase (PH) activity and total lung calcium in SABL treated groups were significantly elevated compared to SASA treated groups starting at 4 days, with PH peaking at 10 days to 163 +/- 13% and calcium peaking at 7 days to 148 +/- 8% of SASA treated groups. The NABL treated animals displayed a significant elevation in PH activity at 4 days only (132 +/- 15%), while the calcium content in this group was significantly increased at 4 and 14 days compared to NASA treated animals. However, the activity of PH in the NABL treated animals was significantly lower than the SABL treated animals at 7, 10, and 14 days. The calcium content of the NABL group was significantly lower than the SABL group at 7 and 10 days. The thiobarbituric acid reactive substance equivalents (TBARS) content and myeloperoxidase (MPO) activity were significantly elevated at all time points in SABL groups as compared to SASA groups, with peak elevation of TBARS to 160 +/- 9% at 4 days and MPO to 268 +/- 40% at 1 day.(ABSTRACT TRUNCATED AT 400 WORDS)