Ventilation-induced changes correlate to pulmonary vascular response and VEGF, VEGFR-1/2, and eNOS expression in the rat model of postnatal hypoxia

Neonatal asphyxia occurs due to reduction in oxygen supply to vital organs in the newborn. Rapid restoration of oxygen to the lungs after a long period of asphyxia can cause lung injury and decline of respiratory function, which result from the activity of molecules that induce vascular changes in the lung such as nitric oxide (NO) and vascular endothelial growth factors (VEGF). In this study, we evaluated the pulmonary and vascular morphometry of rats submitted to the model of neonatal asphyxia and mechanical ventilation, their expression of pulmonary VEGF, VEGF receptors (VEGFR-1/VEGFR-2), and endothelial NO synthase (eNOS). Neonate Sprague-Dawley rats (CEUA #043/2011) were divided into four groups (n=8 each): control (C), control submitted to ventilation (CV), hypoxia (H), and hypoxia submitted to ventilation (HV). The fetuses were harvested at 21.5 days of gestation. The morphometric variables measured were body weight (BW), total lung weight (TLW), left lung weight (LLW), and TLW/BW ratio. Pulmonary vascular measurements, VEGFR-1, VEGFR-2, VEGF, and eNOS immunohistochemistry were performed. The morphometric analysis showed decreased TLW and TLW/BW ratio in HV compared to C and H (P<0.005). Immunohistochemistry showed increased VEGFR-2/VEGF and decreased VEGFR-1 expression in H (P<0.05) and lower eNOS expression in H and HV. Median wall thickness was increased in H, and the expression of VEGFR-1, VEGFR-2, VEGF, and eNOS was altered, especially in neonates undergoing H and HV. These data suggested the occurrence of arteriolar wall changes mediated by NO and VEGF signaling in neonatal hypoxia.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.

A Single Resistance Exercise Session Improves Aortic Endothelial Function in Hypertensive Rats / Uma Única Sessão de Exercício Resistido Melhora a Função Endotelial Aórtica em Ratos Hipertensos

Abstract Background: Physical exercise is an important tool for the improvement of endothelial function. Objective: To assess the effects of acute dynamic resistance exercise on the endothelial function of spontaneously hypertensive rats (SHR). Methods: Ten minutes after exercise, the aorta was removed to evaluate the expression of endothelial nitric oxide synthase (eNOS), phosphorylated endothelial nitric oxide synthase (p-eNOS1177) and inducible nitric oxide synthase (iNOS) and to generate concentration-response curves to acetylcholine (ACh) and to phenylephrine (PHE). The PHE protocol was also performed with damaged endothelium and before and after NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin administration. The maximal response (Emax) and the sensitivity (EC50) to these drugs were evaluated. Results: ACh-induced relaxation increased in the aortic rings of exercised (Ex) rats (Emax= -80 ± 4.6%, p < 0.05) when compared to those of controls (Ct) (Emax = -50 ± 6.8%). The Emax to PHE was decreased following exercise conditions (95 ± 7.9%, p < 0.05) when compared to control conditions (120 ± 4.2%). This response was abolished after L-NAME administration or endothelial damage. In the presence of indomethacin, the aortic rings' reactivity to PHE was decreased in both groups (EC50= Ex -5.9 ± 0.14 vs. Ct -6.6 ± 0.33 log µM, p < 0.05 / Emax = Ex 9.5 ± 2.9 vs. Ct 17 ± 6.2%, p < 0.05). Exercise did not alter the expression of eNOS and iNOS, but increased the level of p-eNOS. Conclusion: A single resistance exercise session improves endothelial function in hypertensive rats. This response seems to be mediated by increased NO production through eNOS activation.

Resumo Fundamento: O exercício físico é uma importante ferramenta para o aprimoramento da função endotelial. Objetivo: Avaliar os efeitos do exercício dinâmico resistido agudo na função endotelial de ratos espontaneamente hipertensos (SHR). Métodos: Após 10 minutos de exercício, a aorta foi removida para avaliação da expressão de óxido nítrico sintase endotelial (eNOS), óxido nítrico sintase endotelial fosforilada (p-eNOS1177) e óxido nítrico sintase endotelial induzível (iNOS), e para a construção de curvas concentração-resposta de acetilcolina (ACT) e fenilefrina (FEN). O protocolo FEN foi também realizado com lesão endotelial e antes e depois da administração de N-nitro-L-arginina metil éster (L-NAME) e indometacina. A resposta máxima (Emax) e a sensibilidade (EC50) a esses fármacos foram avaliadas. Resultados: Houve aumento do relaxamento induzido por ACT nos anéis aórticos dos ratos exercitados (Ex) (Emax = -80 ± 4,6%; p < 0,05) quando comparado àquele dos controles (Ct) (Emax = -50 ± 6,8%). A Emax à FEN diminuiu após exercício (95 ± 7,9%; p < 0,05) quando comparada àquela dos controles (120 ± 4,2%). Tal resposta foi abolida após administração de L-NAME ou lesão endotelial. Na presença de indometacina, a reatividade dos anéis aórticos à FEN diminuiu nos dois grupos (EC50= Ex -5,9 ± 0,14 vs. Ct -6,6 ± 0,33 log µM; p < 0,05/ Emax = Ex 9,5 ± 2,9 vs. Ct 17 ± 6,2%; p < 0,05). O exercício não alterou a expressão de eNOS e de iNOS, mas aumentou o nível de p-eNOS. Conclusão: Uma única sessão de exercício resistido melhora a função endotelial em ratos hipertensos. Essa resposta parece ser mediada por elevação da produção de NO através de ativação de eNOS.

Expression profiles of eNOS, iNOS and microRNA-27b in the corpus cavernosum of rats submitted to chronic alcoholism and Diabetes mellitus

Abstract Purpose: To evaluate the expression of endothelial and inducible NOS in addition to the miRNA-27b in the corpus cavernosum and peripheral blood of healthy rats, diabetic rats, alcoholic rats and rats with both pathologies. Methods: Forty eight Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D) and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study protein expressions of eNOS and iNOS by immunohistochemistry and expression of miRNA-27b in the corpus cavernosum and peripheral blood. Results: Immunohistochemistry for eNOS and iNOS showed an increase in cavernosal smooth muscle cells in the alcoholic, diabetic and alcoholic-diabetic groups when compared with the control group. Similarly, the mRNA levels for eNOS were increased in cavernosal smooth muscle (CSM) in the alcoholic, diabetic and alcoholic-diabetic groups and miRNA-27b were decreased in CSM in the alcoholic, diabetic and alcoholic-diabetic groups. Conclusion: The major new finding of our study was an impairment of relaxation of cavernosal smooth muscle in alcoholic, diabetic, and alcoholic-diabetic rats that involved a decrease in the nitric oxide pathway by endothelium-dependent mechanisms accompanied by a change in the corpus cavernosum contractile sensitivity.

Long-term, regular remote ischemic preconditioning improves endothelial function in patients with coronary heart disease

Remote ischemic preconditioning (RIPre) can prevent myocardial injury. The purpose of this study was to assess the beneficial effects of long-term regular RIPre on human arteries. Forty patients scheduled for coronary artery bypass graft (CABG) surgery were assigned randomly to a RIPre group (n=20) or coronary heart disease (CHD) group (n=20). Twenty patients scheduled for mastectomy were enrolled as a control group. RIPre was achieved by occluding arterial blood flow 5 min with a mercury sphygmomanometer followed by a 5-min reperfusion period, and this was repeated 4 times. The RIPre procedure was repeated 3 times a day for 20 days. In all patients, arterial fragments discarded during surgery were collected to evaluate endothelial function by flow-mediated dilation (FMD), CD34+ monocyte count, and endothelial nitric oxide synthase (eNOS expression). Phosphorylation levels of STAT-3 and Akt were also assayed to explore the underlying mechanisms. Compared with the CHD group, long-term regular RIPre significantly improved FMD after 20 days (8.5±2.4 vs 4.9±4.2%, P<0.05) and significantly reduced troponin after CABG surgery (0.72±0.31 and 1.64±0.19, P<0.05). RIPre activated STAT-3 and increased CD34+ endothelial progenitor cell counts found in arteries. Long-term, regular RIPre improved endothelial function in patients with CHD, possibly due to STAT-3 activation, and this may have led to an increase in endothelial progenitor cells.

Effect of Mesenchymal Stem Cells Associated to Matrixen on the Erectile Function in the Rat Model with Bilateral Cavernous Nerve Crushing Injury

To evaluate the effect of mesenchymal stem cells (MSCs) and MSCs mixed with Matrixen as a cell carrier on the erectile dysfunction caused by bilateral cavernous nerve crushing injury.

White male Sprague-Dawley rats were divided into 4 groups: sham-operated control group (n = 5), bilateral cavernous nerve crushing group (BCNC group, n = 10), BCNC administered with MSCs group (n = 10,1×106 in 20 µL), BCNC administered with Matrixen group (n = 10.1×106 in 20 µL), BCNC administered with MSCs/Matrixen group (n = 10.1×106 in 20 µL). After functional assessment at 4 weeks, major pelvic ganglion (MPG) and penile tissue were collected. Immunofluorescent staining of MPG was performed with PKH26 and Tuj1. Western blot analysis of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were done in corpus cavernosum.

ICP/MAP ratios of BCNC with MSCs and MSCs/Matrixen groups were significantly increased compared with BCNC and BCNC with Matrixen group. Moreover, ICP/MAP ratios of MSCs/Matrixen group were significantly increased compared with BCNC with MSCs group. In MPG, the more implantation of MSCs and increased expression of nerve cells were observed in MSCs/Matrixen group compared with BCNC with MSCs group. Significant increase expression of eNOS and nNOS was also noted in BCNC with MSCs/Matrixen group.

The erectile function was more preserved in MSCs/Matrixen group compared with the administration of MSCs alone in the rats with bilateral cavernous nerve crushing injury. Therefore, we consider that the use of transplant cell carrier such as Matrixen may help the implantation of MSCs and improve the therapeutic effect of MSCs. .

Estrés oxidativo en los músculos soleo y extensor digitorum longus (EDL) de ratas espontáneamente hipertensas / Oxidative stress in soleus and extensor digitorum longus (EDL) muscles of spontaneously hypertensive rats

Las enfermedades sistémicas crónicas afectan el músculo esquelético, siendo la inflamación y el estrés oxidativo algunos de los mecanismos involucrados. El efecto de la hipertensión arterial esencial sobre el músculo esquelético no es bien conocido. Se estudiaron los músculos soleo y extensor digitorum longus (EDL) de ratas espontáneamente hipertensas (SHR), comparadas con las controles normotensas Wistar Kyoto (WKY). Se determinaron los niveles de nitritos y nitratos en µmoles/mg-proteína; las sintasas del óxido nítrico: endotelial (eNOS); neuronal (nNOS); e inducible (iNOS), nitrotirosina y el factor de necrosis tumoral-alfa (TNF-α) en ng/mg-proteína. En las SHR, en el soleo y el EDL respectivamente, se incrementó la nitrotirosina (24,4 ± 5,0 vs. 3,3 ± 0,3, p<0,001; 20,2 ± 4,3 vs. 4,5 ± 0,4, p<0,0037), iNOS (26,6 ± 3,7 vs. 8,3 ± 0,9; 21,3 ± 3,7 vs. 11,0 ± 0,8 ambos p<0,0001), y TNF-α (2,2 ± 0,5 vs. 0,6 ± 0,1, p<0,05; 1,9 ± 0,2 vs. 0,6 ± 0,1, p<0,02); hubo disminución de eNOS en el soleo (20,6 ± 1,4 vs. 30,3 ± 1,2, p<0,00001); de nNOS (soleo 16,8 ± 1,4 vs. 20,7 ± 1,8, p< 0,05; EDL 13,6 ± 1,3 vs. 21,9 ± 1,8, p<0,005) y de nitrito en el EDL (5,8 ± 0,3 vs. 7,1 ± 0,5, p<0,026). En las SHR se observó correlación positiva entre TNF-α vs. nitrotirosina: soleo (r=0,798; p<0,031) y tendencia en EDL (r=0,739; p<0,057); iNOS vs. nitrotirosina (soleo: r=0,908 p<0,0001; EDL: r=0,707; p=0,01), tendencia entre TNF-α vs. iNOS en EDL (r=0,736; p=0,059); y correlación negativa entre eNOS vs. nitrotirosina en soleo (r=-0,816; p=0,0012). En conclusión, las SHR presentan un proceso inflamatorio muscular, evidenciado por el incremento de TNF-α, nitrotirosina, e iNOS. La disminución de las sintasas constitutivas, con incremento de la iNOS es evidencia de la disfunción endotelial.

Systemic diseases affect skeletal muscle, and inflammation and oxidative stress are some of the involved mechanisms. There is scarce information about the effects of essential hypertension on skeletal muscle. The soleus and extensor digitorum longus (EDL) muscles of spontaneously hypertensive rats (SHR) were studied compared to control Wistar Kyoto (WKY) rats. The levels of nitrite and nitrate in µmol/mg-protein; endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) nitric oxide synthases, nitrotyrosine and tumour necrosis factor alpha (TNF-α) in ng/mg-protein were determined. Compared with controls, the SHR showed increased levels of nitrotyrosine (soleus 24.4 ± 5.0 vs. 3.3±0.3, p<0.001; EDL 20.2 ± 4.3 vs. 4.5 ± 0.4, p<0.0037), iNOS (soleus 26.6 ± 3.7 vs. 8.3 ± 0.9; EDL 21.3 ± 3.7 vs. 11.0 ± 0.8, both p<0.0001) and TNF-α (soleus 2.2 ± 0.5 vs. 0.6 ± 0.1, p<0.05; EDL 1.9 ± 0.2 vs. 0.6 ± 0.1, p<0.02). A decrease of eNOS was found in soleus muscle (20.6 ± 1.4 vs. 30.3 ± 1.2, p<0.00001); of nNOS (soleus 16.8 ± 1.4 vs. 20.7 ± 1.8, p< 0.05; EDL 13.6 ± 1.3 vs. 21.9 ± 1.8, p<0.005) and nitrite in EDL (5.8 ± 0.3 vs. 7.1 ± 0.5, p<0.026).There was a positive correlation between TNF-α vs. nitrotyrosine in soleus (r=0.798; p<0.031) and a tendency in EDL (r=0.739; p=0.059); iNOS vs. nitrotyrosine (soleus: r=0.908; p<0.0001; EDL: r=0.707; p<0.01), a tendency between TNF-α and iNOS (EDL: r=0.736; p<0.059); and a negative correlation between eNOS vs. nitrotyrosine in soleus muscle (r=-0.816; p<0.0012). In conclusion, in skeletal muscles of SHR an inflammatory process was found evidenced by the increase in TNF-α, nitrotyrosine and iNOS. The decreased levels of constitutive synthases, together with the higher level of iNOS, are indicative of endothelial dysfunction.

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.