Injection Of MCT Research Articles

Combined physical training protects the left ventricle from structural and functional damages in experimental pulmonary arterial hypertension

BackgroundUnder the adverse remodeling of the right ventricle and interventricular septum in pulmonary arterial hypertension (PAH) the left ventricle (LV) dynamics is impaired. Despite the benefits of combined aerobic and resistance physical trainings to individuals with PAH, its impact on the LV is not fully understood.ObjectiveTo test whether moderate-intensity combined physical training performed during the development of PAH induced by MCT in rats is beneficial to the LV’s structure and function.MethodsMale Wistar rats were divided into two groups: Sedentary Hypertensive Survival (SHS, n = 7); and Exercise Hypertensive Survival (EHS, n = 7) to test survival. To investigate the effects of combined physical training, another group of rats were divided into three groups: Sedentary Control (SC, n = 7); Sedentary Hypertensive (SH, n = 7); and Exercise Hypertensive (EH, n = 7). PAH was induced through an intraperitoneal injection of MCT (60 mg/kg). Echocardiographic evaluations were conducted on the 22nd day after MCT administration. Animals in the EHS and EH groups participated in a combined physical training program, alternating aerobic (treadmill running: 50 min, 60% maximum running speed) and resistance (ladder climbing: 15 climbs with 1 min interval, 60% maximum carrying load) exercises, one session/day, 5 days/week for approximately 4 weeks.ResultsThe physical training increased survival and tolerance to aerobic (i.e., maximum running speed) and resistance (i.e., maximum carrying load) exertions and prevented reductions in ejection fraction and fractional shortening. In addition, the physical training mitigated oxidative stress (i.e., CAT, SOD and MDA) and inhibited adverse LV remodeling (i.e., Collagen, extracellular matrix, and cell dimensions). Moreover, the physical training preserved the amplitude and velocity of contraction and hindered the reductions in the amplitude and velocity of the intracellular Ca2+ transient in LV single myocytes.ConclusionModerate-intensity combined physical training performed during the development of MCT-induced PAH in rats protects their LV from damages to its structure and function and hence increases their tolerance to physical exertion and prolongs their survival.

PS-B04-5: AMELIORATIVE POTENTIAL OF COMBINATION OF VITAMIN D AND SILDENAFIL AGAINST MONOCROTALINE-INDUCED PULMONARY HYPERTENSION IN RATS VIA ACTIVATING ENOS

Objective: The life expectancy in PAH is very less, and only symptomatic treatment is available. Patients with pulmonary hypertension are mostly diagnosed with co-existing vitamin D deficiency. In PAH, there is an increase in the level and expression of PDE 5 enzyme that results in increased degradation of cGMP but there is also a simultaneous decrease in the activation of cGMP due to a subsequent decrease in eNOS mediated nitric oxide synthesis. Thus, limiting the efficacy of sildenafil as monotherapy in the treatment of PAH. Furthermore, vitamin D deficiency is also associated with reduced expression of eNOS mediated NO synthesis, which further decreases cGMP synthesis thus increasing disease severity. Hence, this study was undertaken to study the combined effect of sildenafil and vitamin D therapy in monocrotaline-induced PAH in rats. Design and method: Rats were fed on a vitamin D deficient diet with exposure to filtered fluorescent light to induce vitamin D deficiency. PAH was induced by a single s.c. injection of MCT 50 mg/kg. Rats were divided into 6 groups: Control, MCT, MCT+Sildenafil, MCT+Vitamin D, MCT+Vitamin D+Sildenafil and MCT+L-NAME+Vitamin D+Sildenafil. Haemodynamic, echocardiographic, histopathological and biochemical parameters were analysed. The expression and level of eNOS was done using immunohistochemistry and western blot. Results: The effect of the vitamin D sildenafil combination group was significantly higher than the vitamin D and sildenafil treated alone groups. This protective effect of vitamin D sildenafil combination in PAH is attenuated when administered with L-NAME (20 mg/kg). Furthermore, the administration of MCT reduced the expression of eNOS, which was significantly reversed by the treatment with vitamin D sildenafil combination compared to vitamin D and sildenafil alone. Conclusion: The effect of vitamin D in the treatment of PAH is comparable to sildenafil alone and is further improved when given in combination with sildenafil. Moreover, if pulmonary hypertension is accompanied by vitamin D deficiency it results in increased disease severity of PAH. Thus, sildenafil when combined with vitamin D may exert a synergistic effect in amelioration of symptoms and disease severity in patients of pulmonary hypertension.

Puerarin-V prevents the progression of hypoxia- and monocrotaline-induced pulmonary hypertension in rodent models.

Pulmonary hypertension (PH) is a cardiopulmonary disease characterized by a progressive increase in pulmonary vascular resistance. One of the initial pathogenic factors of PH is pulmonary arterial remodeling under various stimuli. Current marketed drugs against PH mainly relieve symptoms without significant improvement in overall prognosis. Discovering and developing new therapeutic drugs that interfere with vascular remodeling is in urgent need. Puerarin is an isoflavone compound extracted from the root of Kudzu vine, which is widely used in the treatment of cardiovascular diseases. In the present study, we evaluated the efficacy of puerarin in the treatment of experimental PH. PH was induced in rats by a single injection of MCT (50 mg/kg, sc), and in mice by exposure to hypoxia (10% O2) for 14 days. After MCT injection the rats were administered puerarin (10, 30, 100 mg · kg-1 · d-1, i.g.) for 28 days, whereas hypoxia-treated mice were pre-administered puerarin (60 mg · kg-1 · d-1, i.g.) for 7 days. We showed that puerarin administration exerted significant protective effects in both experimental PH rodent models, evidenced by significantly reduced right ventricular systolic pressure (RVSP) and lung injury, improved pulmonary artery blood flow as well as pulmonary vasodilation and contraction function, inhibited inflammatory responses in lung tissues, improved resistance to apoptosis and abnormal proliferation in lung tissues, attenuated right ventricular injury and remodeling, and maintained normal function of the right ventricle. We revealed that MCT and hypoxia treatment significantly downregulated BMPR2/Smad signaling in the lung tissues and PPARγ/PI3K/Akt signaling in the lung tissues and right ventricles, which were restored by puerarin administration. In addition, we showed that a novel crystal type V (Puer-V) exerted better therapeutic effects than the crude form of puerarin (Puer). Furthermore, Puer-V was more efficient than bosentan (a positive control drug) in alleviating the abnormal structural changes and dysfunction of lung tissues and right ventricles. In conclusion, this study provides experimental evidence for developing Puer-V as a novel therapeutic drug to treat PH.

Compound xiebai capsule alleviates pulmonary vascular remodeling in monocrotaline-induced pulmonary arterial hypertension in rats

Purpose: To investigate the effect of compound xiebai capsule (CXC) on pulmonary arterial vascular remodeling in a rat model of monocrotaline-mediated pulmonary arterial hypertension (PAH).Methods: PAH model was established through intraperitoneal injection of MCT (60 mg/kg) to male Sprague-Dawley rats. Thereafter, the rats were grouped and treated with saline (control), nifedipine at a dose of 0.02 g/kg/day (positive control), or different doses of CXC (0.67, 1.00 and 1.32 g/kg/day). The effect of treatments on PAH were determined by measurement of hemodynamic indices and right ventricular hypertrophy index (RVHI). Pathological features and ultrastructure of the lung tissue were examined by H & E staining and transmission electron microscopy (TEM). Immunohistochemistry was employed to determine the expressions of proliferation-associated protein and proliferating cell nuclear antigen (PCNA). Western blotting was applied to assay the expressions of apoptotic pathway proteins.Results: CXC treatment decreased the levels of hemodynamic parameters in PAH rats, alleviated RVHI, improved pulmonary vascular remodeling, decreased vascular wall thickness and area (WT and WA), downregulated the expression of PCNA, and enhanced the expressions of apoptosis-related proteins (cytochrome C, caspase-3, and caspase-9).Conclusion: This is the first study to investigate the effect of CXC on vascular remodeling in PAH. The results indicate that CXC markedly reversed the deleterious changes in PAH-induced rat model.
 Keywords: Pulmonary arterial hypertension, Pulmonary vascular remodeling, Caspase, Cytochrome C, Apoptosis, Compound xiebai capsule

English

English

Alginate Oligosaccharide Alleviates Monocrotaline-Induced Pulmonary Hypertension via Anti-Oxidant and Anti-Inflammation Pathways in Rats.

Pulmonary arterial hypertension (PAH) is a serious and fatal cardiovascular disorder characterized by increased pulmonary vascular resistance and progressive pulmonary vascular remodeling. The underlying pathological mechanisms of PAH are multi-factorial and multi-cellular. Alginate oligosaccharide (AOS), which is produced by depolymerizing alginate, shows better pharmacological activities and beneficial effects. The present study was undertaken to investigate the effects and potential mechanisms of AOS-mediated alleviation of pulmonary hypertension. Pulmonary hypertension was induced in Sprague-Dawley rats by a single intraperitoneal injection of monocrotaline (MCT; 60 mg/kg). Five weeks after the injection of MCT, AOS (5, 10, and 20 mg·kg-1·d-1) was injected intraperitoneally for another three weeks. The results showed that AOS prevented the development of MCT-induced pulmonary hypertension and right ventricular hypertrophy in a dose-dependent manner. AOS treatment also prevented MCT-induced pulmonary vascular remodeling via inhibition of the TGF-β1/p-Smad2 signaling pathway. Furthermore, AOS treatment downregulated the expression of malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase, and pro-inflammatory cytokines, decreased macrophage infiltration, and upregulated the expression of anti-inflammatory cytokines. These findings indicate that AOS exerts anti-oxidative and anti-inflammatory effects in pulmonary arteries, which may contribute to the alleviation of pulmonary hypertension and pulmonary vascular remodeling.

RBMSC/Cav-1F92A Mediates Oxidative Stress in PAH Rat by Regulating SelW/14-3-3η and CA1/Kininogen Signal Transduction.

Background/Objectives Carbonic anhydrase 1 (CA1)/kininogen and selenoprotein W (SelW)/14-3-3η signal transduction orchestrate oxidative stress, which can also be regulated by nitric oxide (NO). The mutated caveolin-1 (Cav-1F92A) gene may enhance NO production. This study explored the effect of Cav-1F92A-modified rat bone marrow mesenchymal stem cells (rBMSC/Cav-1F92A) on oxidative stress regulation through CA1/kininogen and SelW/14-3-3η signal transduction in a rat model of monocrotaline- (MCT-) induced pulmonary arterial hypertension (PAH). Method PAH was induced in rats through the subcutaneous injection of MCT. Next, rBMSC/Vector (negative control), rBMSC/Cav-1, rBMSC/Cav-1F92A, or rBMSC/Cav-1F92A+L-NAME were administered to the rats. Changes in pulmonary hemodynamic and vascular morphometry and oxidative stress levels were evaluated. CA1/kininogen and SelW/14-3-3η signal transduction, endothelial nitric oxide synthase (eNOS) dimerization, and eNOS/NO/sGC/cGMP pathway changes were determined through real-time polymerase chain reaction, Western blot, or immunohistochemical analyses. Results In MCT-induced PAH rats, rBMSC/Cav-1F92A treatment reduced right ventricular systolic pressure, vascular stenosis, and oxidative stress; downregulated CA1/kininogen signal transduction; upregulated SelW/14-3-3η signal transduction; and reactivated the NO pathway. Conclusions In a rat model of MCT-induced PAH, rBMSC/Cav-1F92A reduced oxidative stress by regulating CA1/kininogen and SelW/14-3-3η signal transduction.

Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension

RationaleThe Piezo channel family (Piezo1 and Piezo2), one of the most important family of mechanosensing ion channels. Surprisingly, there are no studies on Piezo in the pulmonary hypertension. We focus on Piezo1 because of its imprtance in vascular deveopment, especially in endothelial cells. Our working hypothesis is that upregulated Piezo1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PAEC proliferation or apoptosis in patients with IPAH.MethodsRNA levels of Piezo1 and Piezo2 was quantified by RT‐PCR Analysis. Protein levels of Piezo1, bax, bcl‐2, Calpain‐1 and Calpain‐2 was quantified by Western blot analysis. adult Male C57BL/6 mice (6–8 weeks) were exposed to persistent chronic hypoxia (10% O2) for 21 days to establish hypoxia‐induced PH. For Sugen 5416/hypoxia (SuHx) induced‐PH rat model, SD rats (age, 6–8 weeks old) were either injected s.c. with 20 mg·kg−1 SU5416 (Sigma‐Aldrich, St. Louis, MO, USA) or vehicle, exposed to hypoxic conditions for 3 weeks (10% O2) and returned to normoxic condition (21% O2) for 2 weeks. In the MCT group, a single intraperitoneal injection of MCT (60 mg/kg, Sigma Aldrich) was given at day 1 to establish the MCT‐induced PH.ResultsPiezo1 and Piezo2 have high expression in rat lung tissue and human PAEC, and also Piezo1 has significantly higher mRNA levels in both human PASMC and PAEC than Piezo2 (n=5, p<0.01). Upregulated Piezo1 in whole‐lung tissue (mainly containing ECs) from MCT‐mediated PH (MCT‐PH) compared to normal control rats (1.00±0.07; 4.74±0.41, n>5, p<0.001). Compared to controls, Piezo1 was increased in whole‐lung tissues in hypoxic mice (1.00±0.07; 4.74±0.41, n>5, p<0.01) and SuHx rats (1.00±0.15; 1.99±0.31, n>5, p<0.01). Also, compared to normoxia condition, increased Piezo1 was associated with a increased Calpain‐1 and Calpain‐2 treated with hypoxia (3% O2 for 72 hours) (1.00±0.16; 4.14±0.75; 1.00±0.14, 2.32±0.35; 1.00±0.07; 1.74±0.14, n>5, p<0.01). Moreover, PAECs from IPAH patients exhibited significant elevation of Piezo1 compared to normal control subjects (1.00±0.09; 6.20±1.78, n=5, p<0.01).ConclusionsThese results are the first to implicate that Piezo1 as a mechanosensor may play an important role in the development of pulmonary hypertension via, at least in part, induction of PAEC apoptosis and enhancement of Ca2+ in PAECs. Piezo1 may be involved in the transcriptional regulation under hypoxia and play critical roles in the development of PH. Therefore, further studies are needed to identify the potential contribution of Piezo1 in PH.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contribution of Mg2+ and Solute Carrier Family 41 to Pulmonary Hypertension in Rat Models

Pulmonary hypertension (PH) is a debilitating disease and characterized by profound vascular remodeling. Many studies indicate that it is related in part to the alterations of Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Mg2+ ion is closely related to Ca2+ ion and is the second‐most abundant divalent cation in animal cells. It is required for the regulation of diverse cellular functions, including cell growth, proliferation, and migration. The Solute Carrier 41 family (SLC41) members including A1, A2 and A3 are Mg2+ transporters, and they have been identified in lung vascular tissue. However, the changes in their expression and functions of SLC41 transporters in PH have not been examined. The present study was designed to investigate whether Mg2+ and its transporters are involved in PH development. PH rats were established by a single injection of MCT or exposed to normobaric hypoxia (CH, 10% O2) for 21 days. Another set of animals received the same treatment were fed with Mg2+ diet supplement (10 mL/kg/d) for 21 days. The expression of SLC41A1/2/3 mRNA and protein in PAs of control and PH rats with or without Mg2+ supplement were determined by Q‐PCR and Western blot. SLC41A1‐2 expressions were significantly increased in PAs of MCT or CH‐treated rats, while mRNA and protein of SLC41A3 were decreased in PH rats. Moreover, PH was attenuated and the changes in SLC41A1‐3 expression were reversed in rats treated with Mg2+ diet supplement. The roles of Mg2+ and SLC41 proteins in PASMCs proliferation and migration were further examined in PASMCs isolated from control rat. They were treated with a control sequence or a SLC41A1/2 targeting siRNA and cultured under low (0.15 mM), normal (1.2 mM) and high (4.8 mM) level of Mg2+. PASMC proliferation monitored using MTS reagent or EdU staining and, migration assessed with Transwell or wound healing assay were markedly inhibited after incubation with high Mg2+ for 48h. Furthermore, siRNA targeting SLC41A1/2 attenuated PASMC proliferation. Our results, hence, clearly show that the SLC41 transporters contribute to PH, and Mg2+ diet supplement could effectively alleviate PH; In addition, Mg2+ and SLC41A1/2 actively participate in PASMC proliferation and migration. These results provide the basis for future study on the roles of Mg2+ and its transporters in PH development.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China NSFC31571179 and NSFC31371165This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 17217: ActRIIA-Fc Rebalances Activin/GDF and BMP9 Signaling to Attenuate Experimental Pulmonary Hypertension

Introduction: Therapies targeting the underlying mechanisms of pulmonary arterial hypertension (PAH) might modify the natural history of disease. The genetics of heritable PAH implicate deficient BMP signaling as a pathogenetic driver. Lung tissues from experimental and human PAH exhibit deficient BMP & excessive TGFβ signaling. While previous studies suggest agonism of BMP9 signaling or inhibition of TGFβ signaling may prevent experimental PH, the roles of activin and growth and differentiation factor (GDF) ligand signaling are unclear. ACTRIIA-Fc (sotatercept) is an activin- and GDF8/11-specific ligand trap currently in Phase 2 studies for anemia and multiple myeloma. Hypothesis: ACTRIIA-Fc inhibits activin and GDF8/11 signaling in vascular cells to attenuate experimental PH and pulmonary vascular remodeling. Methods: Impact of ACTRIIA-Fc on BMP & TGFβ signaling was examined in human pulmonary microvascular endothelial (PMVEC) and pulmonary arterial smooth muscle cells (PASMC). ACTRIIA-Fc (15 mg/kg s.c. 2x/week), sildenafil (30 mg/kg p.o. BID) or vehicle were given prophylactically for 4 weeks to rats developing PH following a single injection of MCT (40 mg/kg s.c.), or via single injection of SU5416 (200 mg/kg s.c.) combined with hypoxia (FIO2=0.10, SU-Hx). Alternately, rats were allowed to develop PH following SU-Hx treatment for 3 weeks or MCT treatment for 4 weeks, and then treated with ACTRIIA-Fc (1, 3, or 10 mg/kg s.c. 2x/week) or isotype-matched control antibody (3 weeks under normoxia following SU-Hx, or 2 weeks following MCT). Invasive hemodynamics, RV mass and vascular remodeling were assessed. Results: In PMVEC, ACTRIIA-Fc enhanced BMP9-induced SMAD1/5/8 activation and transcriptional activity, and reversed inhibition of BMP9 signaling by GDF11. ACTRIIA-Fc inhibited GDF8-, GDF11- and activin A-mediated SMAD2 phosphorylation and myogenic differentiation of PASMC. In vivo , a standard dose of ACTRIIA-Fc prevented PH, RV hypertrophy and PV remodeling in MCT- and SU-Hx-rats, with more potency than sildenafil. All doses of ACTRIIA-Fc attenuated PH and RV hypertrophy when given in a delayed fashion after MCT- and SU-Hx-treatment, with graded effects on PV remodeling. Conclusions: ACTRIIA-Fc rebalances GDF11 vs. BMP9 signaling, with distinct effects in vascular endothelium vs. media, and potently inhibits experimental PH in several rodent models. Given its well-defined tolerability, dosing, and efficacy in previous human studies, ACTRIIA-Fc is an attractive candidate for PAH as a novel, mechanism-targeted therapy.

Involvement of S100A4/Mts1 and associated proteins in the protective effect of fluoxetine against MCT – Induced pulmonary hypertension in rats

BackgroundPulmonary arterial hypertension (PAH) is a complex pulmonary vasculature disease characterized by remodeling of the pulmonary vessels and a persistent increase in the pulmonary vascular resistance (PVR) with a poor prognosis. Serotonin increases the expression of S100A4/Mts1, which in turn stimulates the proliferation and migration of human pulmonary artery smooth muscle cells through the interaction with RAGE (receptor for advanced glycation end products) and thus S100A4/Mts1 has been implicated in the development of PAH in vitro. Fluoxetine, a selective serotonin re-uptake inhibitor has been shown to protect against PAH. The current study was designed to test whether S100A4 and its associated proteins connected in the development of PAH in vivo as well as to investigate the involvement of those proteins in the protective effect of fluoxetine against PAH. MethodsMCT-induced PAH models were established in Wistar rats by a single intraperitoneal injection of MCT (60 mg/kg). Fluoxetine (2 and 10 mg/kg/day) was intragastrically administered once a day for 3 weeks along with controls. The detection methods followed include Hematoxylin and Eosin (H&E) staining, immunohistochemistry, western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR). ResultsMCT induced pulmonary hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy significantly increased the expressions of S100A4 and RAGE in the pulmonary arteries, lungs and right ventricle (RV). Fluoxetine dose-dependently inhibited MCT-induced pulmonary arterial hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy and reduced the S100A4 and RAGE. Further analysis revealed that fluoxetine alleviated both the increase of p53, MMP13, MMP2 and MMP9 and the decrease of pp53Ser15 and MDM2 in lungs and RV tissues of MCT-induced PAH rats. ConclusionFrom the present investigation it could be concluded that S100A4/Mts1 and its associated proteins are involved in the evolution of MCT-induced PAH in rats and fluoxetine inhibits MCT-induced PAH in rats mainly through S100A4/RAGE signaling axis and involved factors.

Baicalein attenuates monocrotaline-induced pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition

AimsEndothelial-to-mesenchymal transition (EndoMT) was shown to lead to endothelial cell (EC) dysfunction in pulmonary arterial hypertension (PAH). Baicalein was reported to inhibit epithelial-to-mesenchymal transition (EMT), a biological process that has many regulatory pathways in common with EndoMT. Whether it can attenuate PAH by inhibiting EndoMT remains obscure. Main methodsPAH was induced by a single subcutaneous injection of MCT (60 mg/kg) in male Sprague Dawley rats. Two weeks after MCT administration, the rats in the treatment groups received baicalein orally (50 or 100 mg/kg/day) for an additional 2 weeks. Hemodynamic changes and right ventricular hypertrophy (RVH) were evaluated on day 28. Cardiopulmonary interstitial fibrosis was detected using Masson's trichrome, Picrosirius-red, and immunohistochemical staining. The reactivity of pulmonary arteries (PAs) was examined ex vivo. The protein expresson of EndoMT molecules, bone morphogenetic protein receptor 2 (BMPR2), and nuclear factor-κB (NF-κB) was examined to explore the mechanism of protective action of baicalein. Key findingsBaicalein (50 and 100 mg/kg) significantly alleviated MCT-induced PAH and cardiopulmonary interstitial fibrosis. Furthermore, baicalein treatment enhanced PA responsiveness to acetylcholine (ACh) in PAH rats. The upregulation of EndoMT molecules (N-cadherin, vimentin, Snail, and Slug) strongly suggest that EndoMT participates in MCT-induced PAH, which was reversed by baicalein (50 and 100 mg/kg) treatment. Moreover, baicalein partially reversed MCT-induced reductions in BMPR2 and NF-κB activation in the PAs. SignificanceBaicalein attenuated MCT-induced PAH in rats by inhibiting EndoMT partially via the NF-κB-BMPR2 pathway. Thus, baicalein might be considered as a promising treatment option for PAH.

Protection against monocrotaline-induced pulmonary arterial hypertension and caveolin-1 downregulation by fluvastatin in rats.

Statins are Hydroxymethylglutaryl-coenzyme A reductase inhibitors, which are typically used to lower blood cholesterol. Additional beneficial effects, including improvement to pulmonary arterial hypertension (PAH), have also been confirmed. However, the mechanisms underlying this improvement have not yet been clarified. The present study was conducted to determine if fluvastatin was protective against experimental PAH development and to investigate the potential effects of fluvastatin on caveolin‑1 (cav‑1) expression. Rats were randomized to either receive a single subcutaneous injection of monocrotaline (MCT; 60mg/kg; MCT group) or a single subcutaneous injection of MCT (60mg/kg) followed by an oral gavage of fluvastatin (10mg/kg) once daily until day 42 (M + F group). Rats in the MCT group received an equivalent volume of saline following the MCT injection. Six additional rats were given an equivalent volume of saline throughout as a control measure. PAH associated variables and cav‑1 protein expression were measured in each group at various times during the experimental period. Hemodynamic and morphometric analysis revealed that M + F rats developed moderate, delayed PAH. Cav‑1 western blot analysis demonstrated that cav‑1 expression was not significantly different in fluvastatin treated rats; however, MCT injured rats given saline had markedly reduced cav‑1 expression. It was concluded that fluvastatin may protect against PAH development and ameliorate MCT induced inhibition of cav‑1 expression in rats.

Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension.

BackgroundPulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans.MethodsPAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured.ResultsMCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration—findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls.ConclusionsProgression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.

Melatonin as a preventive and curative therapy against pulmonaryhypertension.

Pulmonary hypertension (PH) is characterized by elevated pulmonary arterial pressure, which leads to right ventricular (RV) hypertrophy and failure. The pathophysiological mechanisms of PH remain unclear but oxidative stress is believed to contribute to RV dysfunction. Melatonin is a powerful antioxidant and is cardioprotective against ischemia-reperfusion injury and hypertension. Therefore, we hypothesized that a chronic treatment with melatonin, given as a curative or preventive therapy, may confer cardiovascular benefits in PH. PH was induced in Long Evans rats (n≥6 per group), with a single subcutaneous injection of monocrotaline (MCT, 80mg/kg). Melatonin was given daily in the drinking water, with the treatment starting either on the day of the injection of MCT (dose testing: melatonin 75ng/L and 6mg/kg), 14days after the injection of MCT (curative treatment: 6mg/kg), or 5days before the injection (preventive treatment: 6mg/kg). The development of PH was assessed by measuring RV hypertrophy, RV function, cardiac interstitial fibrosis, and plasma oxidative stress. Compared with controls, MCT-treated rats displayed RV hypertrophy and dysfunction, increased interstitial fibrosis, and elevated plasma oxidative stress. A chronic melatonin treatment (75ng/L or 6mg/kg) reduced RV hypertrophy, improved RV function and reduced plasma oxidative stress. Curative and preventive treatment improved RV functional and plasma oxidative stress parameters and reduced cardiac interstitial fibrosis. Our data demonstrate that melatonin confers cardioprotection in this model of PH. As melatonin is an inexpensive and safe drug, we propose that clinical investigation of the effects of melatonin on RV function in patients with PH should be considered.

Glycyrrhizin, inhibitor of high mobility group box-1, attenuates monocrotaline-induced pulmonary hypertension and vascular remodeling in rats.

BackgroundHigh mobility group box-1 (HMGB1), a proinflammatory cytokine, plays a pivotal role in tissue remodeling and angiogenesis, both of which are crucial for the pathogenesis of pulmonary arterial hypertension. In this study, we explored the relationship between HMGB1 and pulmonary hypertension and whether glycyrrhizin, an inhibitor of HMGB1, attenuates disease progression in an animal model of pulmonary hypertension induced by monocrotaline sodium (MCT).MethodsAfter inducing pulmonary hypertension through a single subcutaneous injection of MCT (60 mg/kg) to Sprague–Dawley rats, we administered daily intraperitoneal injections of either glycyrrhizin (GLY, 50 mg/kg), an inhibitor of HMGB1, or saline (control) for either 4 or 6 weeks.ResultsExpression levels of HMGB1 in serum increased from the second week after MCT injection and remained elevated throughout the experiment periods. Lung tissue levels of HMGB1 assessed by immunohistochemical staining at 4 weeks after MCT injection also increased. Chronic inhibition of HMGB1 by GLY treatment reduced the MCT-induced increase in right ventricular (RV) systolic pressure, RV hypertrophy (ratio of RV to [left ventricle + septum]), and pulmonary inflammation. MCT-induced muscularization of the pulmonary artery was also attenuated in the GLY-treated group. As assessed 6 weeks after MCT injection, the GLY-treated group exhibited increased survival (90% [18 of 20]) when compared with the control group (60% [12 of 20]; p =0.0027).ConclusionsGlycyrrhizin, an inhibitor of HMGB1, attenuates pulmonary hypertension progression and pulmonary vascular remodeling in the MCT-induced pulmonary hypertension rat model. Further studies are needed to confirm the potential of HMGB1 as a novel therapeutic target for pulmonary hypertension.

Thermodynamic energy stability and in vitro potassium exchange capacity of ZS-9, a novel selective cation trap for the treatment of hyperkalemia

divided into four subgroups: Group 1: received s.c. injection of phosphate buffered saline; Group 2: received s.c. injection of MCT (MCT alone); Group 3: received injection of MCT followed by i.v. transfusion of rat PAECs transduced with empty adenoviral vector (Null-EC); and Group 4: received injection of MCT followed by i.v. transfusion of rat PAECs overexpressing IL8RA and RB (IL8RA/RB-EC, total 0.5x10 cells/rat). Two days or 4 wks after MCT treatment, eNOS, iNOS, IL-8 (Cinc-2b in rat) and MCP-1 expression and neutrophil and macrophage infiltration in pulmonary arterioles and alveoli, as well as pulmonary arterial flow pattern and arteriole hypertrophy were measured by histological, immunohistochemical staining and echocardiography, respectively. Results: Compared to the MCT alone and MCT-Null-EC groups, transfusion of IL8RA/RB-ECs significantly decreased MCT-induced neutrophil infiltration and pro-inflammatory mediator expression in pulmonary arterioles and alveoli, reduced pulmonary vascular and RV hypertrophy and remodeling. Conclusion: These provocative findings suggest that targeted delivery of PAECs overexpressing IL8RA and IL8RB is effective in repairing injured pulmonary vasculature. Targeted delivery of PAECs to injured lungs provides a novel strategy for the treatment of pulmonary vascular injury in humans.

GW24-e2527 Effects of atorvastatin on BMP/Smad signaling in pulmonary arterial smooth muscle cells derived from pulmonary hypertensive rats

ObjectivesThe present study was to investigate the effects of atorvastatin on BMP/Smad signalling in pulmonary arterial smooth muscle cells (PASMCs) derived from pulmonary artery hypertensive (PAH) rats.MethodsPAH was induced by...

Abstract 5662: Reductions in Atrophic E3 Ubiquitin Ligase Expression in Pulmonary Arteries Correlate With the Development of Pulmonary Hypertension

Vascular remodeling in pulmonary hypertensive syndromes is driven by increased activity of nuclear factor of activated T-cells (NFAT) in smooth muscle cells. Although much is known about the regulation of NFAT activity by calcineurin, very little is known about vascular smooth muscle atrophy F-box ubiquitin E3 ligase expression, which targets calcineurin for proteosomal degradation and inhibits pathological hypertrophy. Therefore, we hypothesized the ubiquitin E3 ligases muscle atrophy F-box (MAFbx) and muscle ring finger (MuRF-1) expression would decline during the development of pulmonary hypertension. To investigate this hypothesis, rats were given a single injection of MCT (50 mg/kg i.p.) and indices of pulmonary hypertension (RVSP and RV/LVS) were measured at 0, 7, 14, 28 and 42 days post MCT injection (n=7–10/group). Left and right pulmonary arteries were harvested from each rat at the conclusion of these measures and mRNA expression was assessed by real-time multiplex PCR analysis for MAFbx and MuRF-1. Consistent with our hypothesis, pulmonary vascular smooth muscle MAFbx and MuRF-1 expression were attenuated from 14d to 42d time points compared to 0d when normalized to b-actin mRNA expression, which correlated negatively with RVSP and right ventricular hypertrophy. Interestingly, while treatment with sildenafil from days 28 – 42 reduced RVSP and RV/LVS, MAFbx expression was further compromised. These results suggest that the E3 ubiquitin ligases MAFbx and MuRF-1 may play a role in the development of MCT-induced pulmonary hypertension.

Rapid Reduction of Liver Steatosis inω3-Depleted Rats Injected with a Novel Lipid Emulsion

The bolus intravenous administration of a novel medium-chain triglyceride: fish oil emulsion (MCT:FO) to normal subjects was recently found to increase within 60 min the amount of long-chain polyunsaturated omega3 fatty acids ( omega3) in platelet and leukocyte phospholipids and, hence, was proposed as a tool to prevent such pathological events as cardiac arrhythmia in selected patients who have to undergo urgent anesthesia and/or surgery. This study investigates whether other cells located outside the vascular bed may also benefit from this procedure for replenishing phospholipids with omega3. For such a purpose, the MCT:FO emulsion (1.0 ml) was injected into normal or omega3-depleted rats examined, one hour later, for the content and fatty acid pattern of liver triglycerides and phospholipids. Control experiments included the administration of saline or a medium-chain triglyceride:olive oil emulsion. The results reveal that the bolus intravenous injection of MCT:FO to the omega3-depleted rats resulted in the enrichment of liver phospholipids in omega3 and a marked reduction in hepatic steatosis. In conclusion, it is proposed that such a procedure may indeed allow a rapid supply of omega3 not only to circulating and vascular endothelial cells but also to extravascular cells, with a resulting correction of the biochemical and biophysical defects linked to a deficiency in these fatty acids.