Inhibition of poly (ADP-ribose) Polymerase-1 (PARP-1) improves endothelial function in pulmonary hypertension.

Endothelial dysfunction is critical in the pulmonary vasculature during pulmonary hypertension (PH). Moreover, in PH, increased inflammation and oxidative/nitrosative stress cause DNA damage, activating poly (ADP-ribose) polymerase-1 (PARP-1). Meloche et al. (2014) and our previous research have shown that inhibiting PARP-1 is protective in PH and associated RV hypertrophy. However, the role of PARP-1 in pulmonary arterial endothelial dysfunction has not been explored completely. Therefore, the current study aims to investigate the involvement of PARP-1 in endothelial dysfunction associated with PH. Hypoxia (1% O2) was used to induce a PH-like phenotype in human pulmonary artery endothelial cells (HPAECs), and PARP-1 inhibition was achieved via siRNA (60 nM). For the in vivo study, male Sprague Dawley rats were administered monocrotaline (MCT; 60 mg/kg, SC, once) to induce PH, and 1, 5-isoquinolinediol (ISO; 3 mg/kg) was administered daily intraperitoneally to inhibit PARP-1. PARP-1 inhibition decreased proliferation and inflammation, as well as improved mitochondrial dysfunction in hypoxic HPAECs. Furthermore, PARP-1 inhibition also promoted apoptosis by increasing DNA damage in hypoxic HPAECs. In addition, inhibition of PARP-1 reduced cell migration, VEGF expression, and tubule formation in hypoxic HPAECs. In in vivo studies, PARP-1 inhibition by ISO significantly decreased the RVP and RVH as well as improved endothelial function by increasing the pulmonary vascular reactivity and expression of p-eNOS in MCT-treated rats.

Reversal of Osteopenia in Ovariectomized Rats by Pentoxifylline: Evidence of Osteogenic and Osteo-Angiogenic Roles of the Drug.

Pentoxifylline (PTX) is a non-selective phosphodiesterase inhibitor and is used for the management of intermittent claudication. We tested whether PTX has oral efficacy in stimulating new bone formation. Rat calvarial osteoblasts (RCO) were used to study the effect of PTX on osteoblast differentiation and angiogenesis. Pharmacokinetic and pharmacodynamic studies were carried out in rats to determine an oral dose of PTX. In ovariectomized (OVX) rats with osteopenia, the effect of PTX on various skeletal parameters was studied, and compared with teriparatide. Effect of PTX on angiogenic signaling was studied by immunoblotting and relevant pharmacologic inhibitors. Bone vascularity was measured by intravenous injection of polystyrene fluorospheres followed by in vivo imaging, and angiogenesis was studied in vitro by tubulogenesis of endothelial cells and in vivo by Matrigel plug assay. Effective concentration (EC50) of PTX in RCO was 8.2 nM and plasma PTX level was 7 nM/mL after single oral dosing of 25 mg/kg, which was 1/6th the clinically used dose. At this dose, PTX enhanced bone regeneration at femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX rats. Furthermore, PTX increased surface referent bone formation parameters and serum bone formation marker (PINP) without affecting the resorption marker (CTX-1). PTX increased the expression of vascular endothelial growth factor and its receptor in bones and osteoblasts. PTX also increased skeletal vascularity, tubulogenesis of endothelial cells and in vivo angiogenesis. Taken together, our study suggested that PTX at 16% of adult human oral dose completely reversed osteopenia in OVX rats by osteogenic and osteo-angiogenic mechanisms.

Injury rate and patterns of Sydney grade cricketers: a prospective study of injuries in 408 cricketers.

BACKGROUND: The grade cricket competition, also known as premier cricket, supplies players to the state and national teams in Australia. The players involved are generally high-performing amateur (subelite) club cricketers. However, to date, there is no study on the injury epidemiology of Australian grade cricket. AIM: To conduct injury surveillance across all teams playing Sydney Grade Cricket (SGC) competition during the 2015-2016 season. METHODS: A cohort study was conducted to track injuries in 408 male cricketers in 20 teams playing SGC competition. Players were tracked through the MyCricket website's scorebook every week. Cricket New South Wales physiotherapists were alerted if there were changes to the playing XI from the last game. If any changes were made due to injury, then an injury incident was registered. RESULTS: During the course of the season, a total of 86 injuries were registered from 65 players, resulting in a loss of 385 weeks of play. The overall injury incidence rate was 35.54 injuries/10 000 playing hours with an average weekly injury prevalence of 4.06%. Lower back injuries (20%) were the most common injuries followed by foot (14%), hand (13.75%), knee (7.5%) and calf (7.5%). Linear regression analysis showed that the likelihood of injury increased as the mean age of the teams increased (R=0.5, p<0.05). CONCLUSION: The injury rate in SGC is lower than that reported at elite level. However, the high rate of lower back injuries (20%) highlights an area of concern in this cohort. High workloads or inadequate physical conditioning may contribute to such injuries. This study sets the foundation for understanding injury epidemiology in grade cricket and examines the links between injury and performance, these results may assist coaches and administrators to develop and implement cricket-specific injury prevention programmes.

Antihypertensive Drugs Aliskiren, Nebivolol, and Olmesartan Reduce Hypertension by Reducing Endothelial Microparticles and Regulating Angiogenesis.

The accelerated generation of endothelial microparticles (EMPs) and impaired angiogenesis are the markers of vascular pathology during various cardiovascular and inflammatory conditions including hypertension. Because studies comparing the effects of antihypertensive agents on these 2 parameters are limited, this study was designed to compare the effects of 3 antihypertensive agents: aliskiren, nebivolol, and olmesartan, on the EMP generation and angiogenesis. Changes in the hemodynamic parameters and serum EMP count were determined after 3 weeks of the drug treatments [aliskiren (30 mg/kg), nebivolol (10 mg/kg), or olmesartan (5 mg/kg) per orally] in L-NAME-induced rat model of hypertension. The 3 drugs prevented the rise in blood pressure and EMP count to a similar extent. Furthermore, nebivolol was found to possess more potent and concentration-dependent antiangiogenic activity compared with aliskiren, whereas olmesartan was devoid of such an effect. The EMPs generated by virtue of the respective drug treatments were found to be involved in mediating the antiangiogenic effect of nebivolol and aliskiren. In addition, olmesartan treatment also resulted in the increased eNOS expression. The results of this study show that the antihypertensive drugs, viz. aliskiren, nebivolol, and olmesartan, regulate the vascular health by their differential effects on the EMP generation and angiogenesis.

Androsin alleviates non-alcoholic fatty liver disease by activating autophagy and attenuating de novo lipogenesis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with therapeutic options on the horizon. Picrorhiza kurroa, enriched with iridoid glycosides like picroside I and picroside II is known for its hepatoprotective activity and anti-inflammatory properties. Androsin, the other phytochemical present in P. kurroa has been shown to have anti-inflammatory and anti-asthmatic properties. However, its role in NAFLD is yet to be investigated. PURPOSE: This study aims to identify the potent hepatoprotective agent from P. kurroa that can attenuate NAFLD in HFrD-fed ApoE-/- mice, and elucidate the underlying mechanisms governing its effects. METHODS: Classical purification methods were used to isolate seven compounds, including picroside I, picroside II and androsin from the roots of P. kurroa. NAFLD-induced ApoE-/- mice were administered orally with either picroside I, picroside II, or androsin for 7 weeks. Animals were scanned non-invasively by ultrasonography at 1st and 14th week. Gross histomorphometry was examined by HE and Sirius red staining. mRNA transcript and protein profile associated with autophagy, lipogenesis, inflammation, and fibrosis was done through RT-PCR and Western blot analysis. RESULTS: In-vitro and in-vivo studies revealed that among the seven evaluated compounds, androsin shows the most potent in-vitro activity. Oral dosing of androsin (10 mg/kg) protected the liver against HFrD-induced NAFLD in ApoE-/- mice model. Biochemical analysis revealed a reduction in ALT and AST enzymes and a significant reduction in cholesterol levels. Hepatocyte ballooning, hepatic lipid deposition, inflammation, and fibrosis were reduced. Androsin treatment significantly reduced fibrosis (α-SMA, collagens, TGF-ß) and inflammation (ILs, TNF-α, NFκB) in ApoE-/- mice. Mechanistically, androsin activated AMPKα and down-regulated the expression of SREBP-1c, resulting in ameliorating hepatic lipogenesis. CONCLUSION: Our results support autophagy as one of the therapeutic strategies to reduce steatosis and hepatic damage. We found that androsin treatment significantly ameliorated hepatic steatosis, serum lipid levels, and hepatic injury in ApoE-/- induced by HFrD. Androsin administration mitigated lipogenesis by inhibiting SREBP1c/FASN pathway and activating autophagy through AMPKα/PI3K/Beclin1/LC3 pathway.

Functionalized azirine based scaffolds as endothelin inhibitors for the selective anti-angiogenic activity.

Anti-angiogenic therapy has long been used as an adjunct therapy for the resolution of tumor burden. The current findings describe the synthesis of novel marine-based azirine-containing compounds that exhibit anti-angiogenic mediated anti-tumor activity. Azirine-2-carboxylate inhibited HUVEC-mediated tubulogenesis without causing cell death in a dose-dependent manner. Ex-vivo CAM, in-vivo Matrigel implantation, and ear angiogenesis experiments have all shown that azirine-2-carboxylate effectively inhibits angiogenesis. Furthermore, azirine-2-carboxylate inhibits the migration of ECs without disrupting the preformed tubule network. Azirine-2-carboxylate had adequate intramuscular systemic exposure and inhibited tumor growth in a xenograft mouse model. DARTS analysis, competitive binding assay, and gene expression investigations revealed that azirine-2-carboxylate inhibits endothelin-1-mediated angiogenesis. Overall, the discovery of azirine-2-carboxylate demonstrated a potent inhibition of angiogenesis targeting ET1 and a possible application in anti-angiogenic therapy.

2,3-Difunctionalized Benzo[b]thiophene Scaffolds Possessing Potent Antiangiogenic Properties.

A new class of 2-anilino-3-cyanobenzo[b]thiophenes (2,3-ACBTs) was studied for its antiangiogenic activity for the first time. One of the 2,3-ACBTs inhibited tubulogenesis in a dose-dependent manner without any toxicity. The 2,3-ACBTs significantly reduced neovascularization in both ex vivo and in vivo angiogenic assays without affecting the proliferation of endothelial cells. Neovascularization was limited through reduced phosphorylation of Akt/Src and depolymerization of f-actin and ß-tubulin filaments, resulting in reduced migration of cells. In addition, the 2,3-ACBT compound disrupted the preformed angiogenic tubules, and docking/competitive binding studies showed that it binds to VEGFR2. Compound 2,3-ACBT had good stability and intramuscular profile, translating in suppressing the tumor angiogenesis induced in a xenograft model. Overall, the present study suggests that 2,3-ACBT arrests angiogenesis by regulating the Akt/Src signaling pathway and deranging cytoskeletal filaments of endothelial cells.

Involvement of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) in endothelial dysfunction associated with pulmonary hypertension.

AIMS: Increased proliferation, inflammation, and endothelial microparticle (EMP) generation in the pulmonary vasculature lead to endothelial dysfunction in pulmonary hypertension (PH). Interestingly, MK2, a downstream of p38MAPK, is a central regulator of inflammation, proliferation, and EMP generation in cardiovascular diseases. However, the role of MK2 in pulmonary endothelial dysfunction remains unexplored. MAIN METHODS: The Human Pulmonary Artery Endothelial cells (HPAECs) were exposed to hypoxia (1% O2) for 72 h, and MK2 inhibition was achieved by siRNA treatment. Western blotting, qualitative RT-PCR, immunocytochemistry, flow cytometry and enzyme-linked immunoassays were conducted to study pathological alterations and molecular mechanisms. Neoangiogenesis was studied using cell migration and tubule formation assays. For in vivo study, Male Sprague Dawley rats and MK2 knock-out mice with littermate control were treated with monocrotaline (MCT) 60 mg/kg and 600 mg/kg, respectively (s.c. once in rat and weekly in mice) to induce PH. MMI-0100 (40 µg/kg, i.p. daily for 35 days), was administered in rats to inhibit MK2. KEY FINDINGS: MK2 inhibition significantly decreased inflammation, cell proliferation, apoptosis resistance, and improved mitochondrial functions in hypoxic HPAECs. Hypoxia promoted cell migration, VEGF expression, and angiogenesis in HPAECs, which were also reversed by MK2 siRNA. MK2 inhibition decreased EMP generation and increased the expression of p-eNOS in hypoxic HPAECs, a marker of endothelial function. Furthermore, MK2 deficiency and inhibition both reduced the EMP generation in mice and rats, respectively. SIGNIFICANCE: These findings proved that MK2 is involved in endothelial dysfunction, and its inhibition may be beneficial for endothelial function in PH.

Synthesis and Evaluation of a Zinc Eluting rGO/Hydroxyapatite Nanocomposite Optimized for Bone Augmentation.

Repair of critical size bone defects is a clinical challenge that usually necessitates the use of bone substitutes. For successful bone repair, the substitute should possess osteoconductive, osteoinductive, and vascularization potential, with the ability to control post-implantation infection serving as an additional advantage. With an aim to develop one such substitute, we optimized a zinc-doped hydroxyapatite (HapZ) nanocomposite decorated on reduced graphene oxide (rGO), termed as G3HapZ, and demonstrated its potential to augment the bone repair. The biocompatible composite displayed its osteoconductive potential in biomineralization studies, and its osteoinductive property was confirmed by its ability to induce mesenchymal stem cell (MSC) differentiation to osteogenic lineage assessed by in vitro mineralization (Alizarin red staining) and expression of osteogenic markers including runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP), type 1 collagen (COL1), bone morphogenic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). While the potential of G3HapZ to support vascularization was displayed by its ability to induce endothelial cell migration, attachment, and proliferation, its antimicrobial activity was confirmed using S. aureus. Biocompatibility of G3HapZ was demonstrated by its ability to induce bone regeneration and neovascularization in vivo. These results suggest that G3HapZ nanocomposites can be exploited for a range of strategies in developing orthopedic bone grafts to accelerate bone regeneration.

Fatty acid synthase modulates proliferation, metabolic functions and angiogenesis in hypoxic pulmonary artery endothelial cells.

Endothelial dysfunction plays an important role in structural remodeling occurring in the pulmonary vasculature during pulmonary hypertension (PH). Endothelial injury causes apoptosis and activation of endothelial cells. However, some endothelial cells show apoptosis-resistance and later proliferate extensively leading to vascular oculopathy and formation of plexiform lesions in PH. Studies have shown that rapidly proliferating cells exhibit increased expression of Fatty acid synthase (FAS), a regulatory enzyme responsible for the production of fatty acids. Our previous study has shown that FAS inhibition prevented smooth muscle cell proliferation, reversed pulmonary vascular remodeling and improved pulmonary vasoreactivity in monocrotaline induced PH model. However, the role of FAS in pulmonary artery endothelial cell proliferation and angiogenesis has not been explored. The present study was designed to explore the role of FAS in proliferation, metabolic dysfunctions, and angiogenesis in endothelial dysfunction associated with PH. The human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia and FAS siRNA (60nM) was used for the FAS inhibition. Increased expression and activity of FAS were observed in hypoxic HPAECs. Inhibition of FAS increased apoptosis and glucose oxidation, but decreased cellular proliferation, markers of autophagy and glycolysis in hypoxic HPAECs. FAS inhibition decreased the angiogenesis as evident by decreased tubule length and VEGF expression in hypoxic HPAECs. Inhibition of FAS also increased expression of endothelial NOS in hypoxic HPAECs, a marker of endothelial function. Our results proved, and further supported previous findings, that inhibition of FAS is beneficial for endothelial function in pulmonary hypertension.

An overview of geospatial methods used in unintentional injury epidemiology.

BACKGROUND: Injuries are a leading cause of death and disability around the world. Injury incidence is often associated with socio-economic and physical environmental factors. The application of geospatial methods has been recognised as important to gain greater understanding of the complex nature of injury and the associated diverse range of geographically-diverse risk factors. Therefore, the aim of this paper is to provide an overview of geospatial methods applied in unintentional injury epidemiological studies. METHODS: Nine electronic databases were searched for papers published in 2000-2015, inclusive. Included were papers reporting unintentional injuries using geospatial methods for one or more categories of spatial epidemiological methods (mapping; clustering/cluster detection; and ecological analysis). Results describe the included injury cause categories, types of data and details relating to the applied geospatial methods. RESULTS: From over 6,000 articles, 67 studies met all inclusion criteria. The major categories of injury data reported with geospatial methods were road traffic (n = 36), falls (n = 11), burns (n = 9), drowning (n = 4), and others (n = 7). Grouped by categories, mapping was the most frequently used method, with 62 (93%) studies applying this approach independently or in conjunction with other geospatial methods. Clustering/cluster detection methods were less common, applied in 27 (40%) studies. Three studies (4%) applied spatial regression methods (one study using a conditional autoregressive model and two studies using geographically weighted regression) to examine the relationship between injury incidence (drowning, road deaths) with aggregated data in relation to explanatory factors (socio-economic and environmental). CONCLUSION: The number of studies using geospatial methods to investigate unintentional injuries has increased over recent years. While the majority of studies have focused on road traffic injuries, other injury cause categories, particularly falls and burns, have also demonstrated the application of these methods. Geospatial investigations of injury have largely been limited to mapping of data to visualise spatial structures. Use of more sophisticated approaches will help to understand a broader range of spatial risk factors, which remain under-explored when using traditional epidemiological approaches.