Intrapulmonary Arteries Research Articles

Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension

The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice (Bmp4(LacZ/+)), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 (Bmp2(+/-)) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2(+/-) but not Bmp4(LacZ/+) mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.

Abstract 4923: Prostacyclin Analogues Activates Calcium-dependent Potassium (K Ca ) Channels via PPAR β / δ in Pulmonary Artery

Pulmonary hypertension is associated with vasoconstriction, thrombosis and proliferation, and the lack of endogenous prostacyclin. Prostacyclin and its analogues are potent vasodilators, representing an important pillar of modern therapy of this devastating disease. Potassium channels determine the membrane potential of pulmonary artery smooth muscle cells (PASMCs) and are important controllers of their calcium homeostasis and consequently the pulmonary arterial tone. In the present study, we investigated the signaling pathway and effects of the stable prostacyclin analogues treprostinil and iloprost in primary human PASMCs and isolated rat pulmonary artery. We employed the whole-cell patch-clamp technique combined with small interfering (si)RNA and live-cell calcium imaging on human PASMCs and the myograph-mounted isolated intrapulmonary artery model. We found that treprostnil (10 nM) and iloprost (10 nM) hyperpolarized primary human PASMCs (13.1 ± 2.2 mV, p < 0.01 and 15.7 ± 2 mV, p < 0.01 respectively, n = 6 for each group) and activated K Ca (IC 50 = 0.2 ± 0.05 nM and IC 50 = 0.5 ± 0.2 nM respectively, n = 5 for each group) at clinically relevant concentrations. The IP receptor antagonist RO1138452 (10 μ M) and PKA blocker KT5720 (1 mM) abolished these effects. In the siRNA-transfected cells no enhancement of K Ca current was detected. The novel PPAR β / δ antagonist GSK0660 (10 nM), abolished the activation of K Ca current by iloprost or treprostinil, whereas the selective PPAR β / δ agonist GW0742 (10 nM) activated the K Ca current. Calcium imaging measurements supported these data showing that GW0742 reduced the basal intracellular calcium concentration by 9.41±2.99 % (n = 25, p < 0.01) compared to the baseline in human PASMCs. Consequently, GW0742 caused relaxation in U46619 -preconstricted rat intrapulmonary arteries (IC 50 = 9.42 μ M, n = 5). This is the first study implicating K Ca in the prostacyclin analogue- mediated hyperpolarization via PPAR β / δ in human PASMCs. This might represent a novel pathway related to the vasodilating effects of prostanoids.

Lung Tissue Blood Perfusion Changes Induced by in utero Tracheal Occlusion in a Rabbit Model of Congenital Diaphragmatic Hernia

Objective: To analyze the impact of in utero tracheal occlusion (TO) on lung tissue blood perfusion, as measured by fractional moving blood volume (FMBV) and conventional spectral Doppler, in a rabbit model of congenital diaphragmatic hernia (CDH). Methods: In 50 fetal rabbits, a left CDH was surgically created at 23 days of gestational age (GA). At 28 days of GA, the surviving CDH fetuses were randomly assigned to undergo either TO (CDH+TO group) or a sham operation (CDH group). Twenty littermates, which were not operated on, served as internal normal controls. At 30 days of GA, lung perfusion estimated by FMBV and spectral Doppler of the proximal intrapulmonary artery were evaluated in the right lung during cesarean section. Doppler waveform analysis included the pulsatility index (PI), peak early diastolic reverse flow and peak systolic velocity. Results: Eleven CDH fetuses, 9 CDH+TO and 20 controls were suitable for the study. CDH fetuses showed a significantly higher PI [8.0 (SD 1.8) vs. 5.22 (SD 1.1), p < 0.001] and lower FMBV [13.5% (SD 4.6) vs. 23.0% (SD 2.1), p < 0.001] than the controls. In contrast, CDH+TO fetuses had a significantly lower PI [5.8 (SD 2.3) vs. 8.0 (SD 1.8), p = 0.015] and higher FMBV [27.6% (SD 7.1) vs. 13.5% (SD 4.6), p < 0.001] than CDH fetuses, with values similar to the controls. Peak early diastolic reverse flow and peak systolic velocity showed nonsignificant differences among the study groups. The lung to body weight ratio at necropsy correlated positively with lung FMBV (r = 0.60, p < 0.001) and negatively with the pulmonary artery PI (r = –0.48, p < 0.01). Conclusion: Tracheal occlusion is consistently associated with increased lung tissue perfusion and decreased intrapulmonary impedance in a rabbit model of CDH.

Organ culture mimics the effects of hypoxia on membrane potential, K+ channels and vessel tone in pulmonary artery

Blood vessel culture is gaining interest for use with transfection-based techniques, but alters the contractile properties of the vessels. The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K(+) channels regulating the resting membrane potential (E(m)) and tone of pulmonary artery smooth muscle cells (PASMCs). Rat intrapulmonary arteries were isolated and cultured under standard and modified conditions. Contractile responses of fresh and cultured PA were compared using vessel myograph. Electrophysiology experiments on isolated PASMCs used the patch-clamp technique. K(+) channel expression was quantified using reverse transcription and real-time PCR. After 4 days in culture vessels contracted to phenylephrine, but relaxation to carbachol was significantly impaired. Contractile responses to 10 mM KCl, 4-aminopyridine and tetraethylammonium increased, and vessels developed an uncharacteristic relaxation response to Ca(2+)-free solution, nifedipine and levcromakalim. PASMCs from cultured vessels were depolarized and K(+) currents reduced, in association with down-regulation of K(v)1.5, K(v)2.1 and TWIK-related acid-sensitive K(+) channel-1 mRNA. These changes were partially reversed by increased oxygenation of the culture medium or removing the endothelium before culture. Culture of PA for 3-4 days induced loss of functional K(+) channels, depolarization of PASMCs, Ca(2+) influx, intrinsic tone and spontaneous constrictions, similar to the effects of chronic hypoxia. This limits the use of cultured vessels for studying excitation-contraction coupling, although oxygenating the culture medium and removing the endothelium can help to retain normal smooth muscle function.

Expression of 15-lipoxygenases in pulmonary arteries after hypoxia

We have previously reported that subacute hypoxia upregulates and activates the steady state expression of pulmonary artery 15-lipoxygenase (15-LOX). However, there are two 15-LOX isoenzymes, 15-LOX-1 and 15-LOX-2, which are expressed in pulmonary arteries that have not been previously studied. In the present study, we identified the differential expression of the two isoenzymes in normoxic or hypoxic intrapulmonary arteries of rats, pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) from the main branches of pulmonary artery of neonatal bovines. Immunohistochemistry, in situ hybridisation, RT-PCR, and Western blotting were employed to identify the differential expression of the two isoenzymes. 15-LOX-1 was expressed in both normoxic and hypoxic rat arteries. In contrast, 15-LOX-2 was hardly detectable in normoxic vessels by immunohistochemistry and Western blotting, but it was detected in hypoxic arteries. In cultured bovine pulmonary artery cells, 15-LOX-1 and 15-LOX-2 were found to be different in cellular distribution. The expression of 15-LOX-1 was stronger in PAECs than in PASMCs, whereas, 15-LOX-2 was weaker in PAECs than in PASMCs. Moreover, the expressions of both 15-LOX isoenzymes increased dramatically after hypoxia. These results suggest that both isoenzymes of 15-LOXs are upregulated after hypoxia.

Vascular Smooth Muscle Modulates Endothelial Control of Vasoreactivity via Reactive Oxygen Species Production through Myoendothelial Communications

BackgroundEndothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC)/smooth muscle cells (SMC) crosstalk is far from complete. Here, we have examined the role of gap junctions and reactive oxygen species (ROS) in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone.Methodology/Principal FindingsIn small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses and immunofluorescent labeling evidenced connexin (Cx) 37, 40 and 43 in EC and/or SMC. Functional experiments showed that the Cx-mimetic peptide targeted against Cx 37 and Cx 43 (37,43Gap27) (1) reduced contractile and calcium responses to serotonin (5-HT) simultaneously recorded in pulmonary arteries and (2) abolished the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolise superoxide anion and H2O2, respectively. Both Cx- and ROS-mediated effects on the responses to 5-HT were reversed by L-NAME, a NO synthase inhibitor or endothelium removal. Electronic paramagnetic resonance directly demonstrated that 5-HT-induced superoxide anion production originated from the SMC. Finally, whereas 5-HT increased NO production, it also decreased cyclic GMP content in isolated intact arteries.Conclusions/SignificanceThese data demonstrate that agonist-induced ROS production in SMC targeting EC via myoendothelial gap junctions reduces endothelial NO-dependent control of pulmonary vasoreactivity. Such SMC modulation of endothelial control may represent a signaling pathway controlling vasoreactivity under not only physiological but also pathological conditions that often implicate excessive ROS production.

MEASUREMENT OF PULMONARY FLOW RESERVE IN HIGHER PRIMATES

1. There are currently limited diagnostic methods for assessing the integrity of the pulmonary microvasculature. We hypothesized that a novel, invasively determined physiological index of 'pulmonary flow reserve' (PFR = maximal hyperaemic pulmonary blood flow divided by basal pulmonary flow) may facilitate microvascular assessment in the lung. Therefore, we developed a baboon model in which to: (i) validate the use of Doppler flow velocity for PFR assessment; (ii) define the optimal drug and dose regimen for attainment of maximal pulmonary hyperaemia; and (iii) demonstrate the feasibility of measuring PFR in healthy higher primates. 2. Doppler sensor guidewires were placed in segmental pulmonary arteries of 11 ketamine-anaesthetized baboons. Vessel diameter, flow velocity and haemodynamics were recorded before and after direct intrapulmonary artery administration of saline, adenosine (50-500 microg/kg per min) and papaverine (3-60 mg), enabling calculation of PFR. 3. Saline (either bolus injection or infusion) did not alter vessel diameter or flow velocity (P > 0.1), validating local drug administration. Both adenosine and papaverine induced dose-dependent increases in flow velocity from baseline (from 22.5 +/- 2.3 to 32.7 +/- 4.8 cm/s for 400-500 microg/kg per min adenosine; and from 23.9 +/- 1.1 to 34.6 +/- 4.0 cm/s for 24 mg papaverine; both P < 0.0001), without affecting pulmonary artery pressure or vessel diameter (P > 0.3). Healthy primate PFR values were 1.35 +/- 0.10 and 1.39 +/- 0.10 using 200 microg/kg per min adenosine and 24 mg papaverine, respectively (P > 0.8). 4. In conclusion, pulmonary flow reserve in higher primates can be assessed using Doppler sensor guidewire and either adenosine or papaverine as microvascular hyperaemic agents. Measurements of PFR may facilitate pulmonary microvascular assessments.

Senescent endothelial progenitor cells from dogs with pulmonary arterial hypertension: a before-after self-controlled study.

Previous studies have underlined the importance of endothelial dysfunction and microvascular occlusion in the pathogenesis of pulmonary artery hypertension (PAH). Since the endothelial progenitor cells (EPCs) are involved in maintaining endothelial homeostasis, we observed the change of peripheral EPCs in canines before and after PAH onset. PAH was induced by intra-pulmonary artery injection of dehydromonocrotaline (DHMC) in nine beagles. Before and 48 h and 6 weeks after DHMC injection, 40 ml peripheral blood was obtained from the femoral vein. Circulating EPCs were identified as CD133 + KDR + cells and numerated by fluorescence-activated cell sorter; the EPCs functional capacity was determined by in vitro tubule-forming assay. The senescence of EPCs was determined by beta-galactosidase staining. At each time point, 2 ml blood from femoral artery was obtained for arterial oxygen pressure (PaO(2)). Forty-eight hours after DHMC injection, treated beagles suffered from hypoxemia; however, both the number and the tubule-forming capacity of EPCs were transiently raised. Six weeks later, PAH was confirmed by obviously high mean pulmonary arterial pressure (20.2 +/- 1.64 vs. 11.3 +/- 2.0 mmHg, p < 0.05) and low PaO(2) (69.30 +/- 9.15 vs. 95.94 +/- 1.43 mmHg, p < 0.01) in beagles after DHMC treatment, and their EPCs exhibited a predominant decrease in either the number (206.1 +/- 26.8 vs. 632.8 +/- 42.8 cells/ml blood, p < 0.01) or the tubule-forming capacity (21.1 +/- 2.8 vs. 11.2 +/- 2.8 tubules/x200 field, p < 0.01). Additionally, senescence-associated beta-galactosidase-positive EPCs were significantly increased. Our data suggested that, after the acute stage of DHMC injury to pulmonary vessels, the EPCs from PAH beagles suffered from exhaustion and senescence.

Pulmonary apelin levels and effects in rats with hypoxic pulmonary hypertension

The peptide apelin is localised in the vascular endothelium and highly expressed in pulmonary tissue. The aim of this study was to investigate whether apelin could be a potential lung-derived plasma marker for pulmonary hypertension, and study the effect of apelin in pulmonary arteries. Apelin protein levels were measured in the lung, right ventricle, and plasma from normoxic and chronic hypoxic rats with pulmonary hypertension. Isolated intrapulmonary arteries were mounted in microvascular myographs and the effect of apelin investigated. Finally, the distribution of apelin receptors in pulmonary tissue was visualised by immunohistochemistry. Total pulmonary apelin content was not changed by hypoxia. Right ventricular apelin concentrations and content were lower than in the lung, but increased substantially in hypoxia in correlation with right ventricular pressure. Plasma apelin did not reflect pulmonary or right ventricular apelin levels. In pulmonary arteries from normoxic rats, apelin inhibited vasoconstriction to endothelin-1 and angiotensin-II. However, in arteries from hypoxic rats, apelin failed to inhibit contraction to angiotensin-II and endothelin-1. No difference in immunoreaction for apelin receptors was found in lung sections and arteries from normoxic versus chronic hypoxic rats. Apelin changes in the right ventricle seem more specific for pulmonary hypertension than do changes in pulmonary tissue, which does not speak in favour of apelin as a lung-derived marker for this disease. During normoxic conditions, apelin has a modulating effect on vasoconstriction which is lost in chronic hypoxia. This may reflect alterations in the signal transduction downstream of the apelin receptor.

NK 1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK 1 receptors, and endothelium-independent contraction (EIC) via tachykinin NK 2 receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK 1 receptor. Five tachykinin agonists, including septide, a reportedly atypical NK 1 agonist, caused concentration-dependent EDR in the presence of NK 2 antagonist (SR-48968) + TXA 2 synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK 2 antagonist (SR-48968) + nitric oxide synthase inhibitor ( l-N G-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC 50 values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK 1 agonists to NK 1 receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala 5, β-Ala 8]-α-neurokinin fragment 4–10 (NK 2 agonist) and senktide (NK 3 agonist) caused no responses. Two structurally different NK 1 antagonists, CP-99994 and SR-140333, shifted the concentration–EDC and –EDR curves of SPME, a selective NK 1 agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK 1 antagonists to NK 1 receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca 2+ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca 2+ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK 1 receptor followed by an increase in intracellular Ca 2+, and sensitivity to Ca 2+ may be higher in the EDR than EDC pathway.

Vascular smooth muscle cells limit endothelial control of vasoreactivity via ROS production through myoendothelial communications

Although cross‐talk between endothelial (EC) and smooth muscle cells (SMC) through myoendothelial gap junctions play a major role in the vasoreactivity, the transduction pathways remain unclear. Here we tested the hypothesis of the role of ROS and gap junctions in this cross‐talk.Connexin (Cx)‐ mimetic peptide targeted against Cx 37 and Cx 43 (37,43Gap27) reduced (1) the contractile and calcium responses to serotonin (5‐HT) simultaneously recorded in small intrapulmonary arteries and (2) abolished the diffusion, in the SMC, of carboxyfluorescein‐AM loaded in EC. Quantitative RT‐PCR, Western Blot analyses and immunofluorescent labeling showed the presence of Cx 37, 40 and 43. Superoxyde dismutase and catalase which destroy superoxyde anion and H2O2 respectively also decreased the contractile and calcium responses to 5‐HT. Both Cx‐ and ROS‐mediated effects on the responses to 5‐HT were reversed by L‐NAME and/or endothelium removal thus reflecting a cross‐talk between SMC and EC through ROS and gap junctions. Electronic paramagnetic resonance demonstrated that superoxyde anion production by 5‐HT originated from the SMC only. Moreover, 5‐HT decreased cyclic GMP content in isolated arteries.These data support a model where superoxyde anion produced by 5‐HT in the SMC will pass through myoendothelial gap junctions to reduce endothelial NO‐dependent control of vasoreactivity.

Reduced basal Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats is restored by administration of the scaffolding domain of caveolin‐1

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with impaired endothelial Ca2+ influx in response to store depletion. Molecular candidates for store‐operated and receptor operated Ca2+ entry (SOCE/ROCE) such as transient receptor potential (TRP) channels may also be involved in basal influx and appear to be trafficked to the plasma membrane by caveolin‐1 (cav‐1). We hypothesized that impaired basal Ca2+ entry occurs following CH and is due to deficient cav‐1 mediated trafficking of relevant ion channels. Ca2+ influx was assessed in isolated pressurized intrapulmonary arteries from control and CH rats (4 weeks at 0.5 atm) by Mn2+ quenching of fura‐2 fluorescence in the presence of a cav‐1 scaffolding domain peptide (AP‐CAV) or scrambled control. AP‐CAV increased basal Ca2+ influx in arteries from CH animals, and had no effect in the controls. In additional studies, we observed reduced immunofluorescent membrane localization of TRPC4 and cav‐1 in cultured bovine pulmonary artery endothelial cells (BPAECS) exposed to hypoxia (2% O2, 4 days) compared to controls. These studies suggest that hypoxia per se impairs cav‐1 mediated trafficking of ion channels that may be responsible for basal endothelial Ca2+ influx.

The shy Angeli and his elusive creature: the HNO route to vasodilation

what we now call Angeli's salt (AS) was discovered more than a century ago by the Italian chemist Angelo Angeli (Fig. 1) . He found that “salts of nitroxylaminic acid are readily resolved into the corresponding nitrites and the unsaturated residue nitroxyl” (HNO), proposing their use as suppliers of HNO (3). However, by analogy with the genial but very reclusive personality of the discoverer (17), the physiological properties of HNO didn't draw too much attention until early 1990s. In their seminal work, Fukuto and colleagues (19) reported that HNO elicits vasorelaxation in both the rabbit aorta and bovine intrapulmonary artery by a soluble guanylate cyclase (sGC)- dependent pathway. Later, it was evident that the thiol-donating agent l-cysteine can discriminate the vasodilatative profile of HNO from that of nitric oxide (NO·) or nitrosothiols: AS/HNO action can be blocked by this agent, whereas the NO· effect is potentiated (36). In combination with the suggestion that NO· is the only species able to directly activate sGC (9), these data led to the conclusion that HNO-induced vasorelaxation is due to its intracellular conversion to NO· with subsequent activation of sGC. For several years, this idea has relegated HNO to a mere precursor of the presence and biological activity of NO·, de facto negating to HNO its own right to exist.

J008 Vascular smooth muscle modulates endothelial control of vasoreactivity via ros production through myoendothelial communications

Endothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC) / smooth muscle cells (SMC) crosstalk is far from complete. Here, we have examined the role of gap junctions in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone. In small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses revealed the presence of 3 connexins (Cx): 37, 40 and 43. Immunofluorescence labelling showed the presence of Cx 37, 40 and 43 in EC and the presence of Cx 37 and 40 in SMC. Functional experiments showed that the peptides homologous to the gap 26 and 27 domains of the extracellular loops of Cx 43 (43Gap 26) or Cx 37 and 43 (37-43Gap 27) respectively, (1) interrupt intercellular communications in a specific manner, (2) attenuate the calcium and contractile responses to serotonin (5-HT) simultaneously recorded in pulmonary arteries and (3) abolish the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolize superoxide anion (O2•-) and H2O2, respectively. Both Cx- and reactive oxygen species (ROS)-mediated effects on the responses to 5-HT were reversed by (1) L-NAME, a NO synthase inhibitor, or (2) endothelium removal with CHAPS. Electron paramagnetic resonance directly demonstrated that 5-HTinduced ROS production, and more specifically O2•-, originated from the SMC. Finally, 5-HT decreased basal cyclic GMP content in isolated intact arteries, whereas a cocktail of 5-HT, superoxide dismutase and catalase increased basal cyclic GMP content. Altogether, the findings suggest that 5-HT produces O2•- in the smooth muscle and NO in the endothelium. O2•- passes through the myoendothelial junctions to decrease endothelial NO production and thus strengthen pulmonary vasoreactivity. This is the first time that we demonstrate a control of the endothelial NO function by the smooth muscle is demonstrated. Since ROS is often overproduced in many cardiovascular diseases, such process could also be of great importance in pathological conditions.

79. Increased Recruitment and Proliferation of Intrapulmonary Arterial Perivascular Precursor Cells in Experimental Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is associated with specific pulmonary vascular abnormalities including decreased pulmonary arterial branching, increased muscularization of intrapulmonary arteries, and more distal muscularization within the pulmonary arterial tree. These abnormalities result in varying degrees of pulmonary hypertension, which along with lung hypoplasia, contribute to respiratory distress in neonates with CDH. The process of vascular wall maturation is dependent upon cell signaling interactions between endothelial cells of nascent vessels and the surrounding mesenchyme that lead to the recruitment of perivascular progenitor cells to endothelium tubes and subsequent proliferation and differentiation of these cells to form the vascular smooth muscle cells and pericytes of the arterial wall.

Effects of streptozotocin-induced diabetes on the pharmacology of rat conduit and resistance intrapulmonary arteries

BackgroundPoor control of blood glucose in diabetes is known to promote vascular dysfunction and hypertension. Diabetes was recently shown to be linked to an increased prevalence of pulmonary hypertension. The aim of this study was to determine how the pharmacological reactivity of intrapulmonary arteries is altered in a rat model of diabetes.MethodsDiabetes was induced in rats by the β-cell toxin, streptozotocin (STZ, 60 mg/kg), and isolated conduit and resistance intrapulmonary arteries studied 3–4 months later. Isometric tension responses to the vasoconstrictors phenylephrine, serotonin and PGF2α, and the vasodilators carbachol and glyceryl trinitrate, were compared in STZ-treated rats and age-matched controls.ResultsSTZ-induced diabetes significantly blunted the maximum response of conduit, but not resistance pulmonary arteries to phenylephrine and serotonin, without a change in pEC50. Agonist responses were differentially reduced, with serotonin (46% smaller) affected more than phenylephrine (32% smaller) and responses to PGF2α unaltered. Vasoconstriction caused by K+-induced depolarisation remained normal in diabetic rats. Endothelium-dependent dilation to carbachol and endothelium-independent dilation to glyceryl trinitrate were also unaffected.ConclusionThe small resistance pulmonary arteries are relatively resistant to STZ-induced diabetes. The impaired constrictor responsiveness of conduit vessels was agonist dependent, suggesting possible loss of receptor expression or function. The observed effects cannot account for pulmonary hypertension in diabetes, rather the impaired reactivity to vasoconstrictors would counteract the development of pulmonary hypertensive disease.

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca2+ ([Ca2+]i), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC20) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF2α-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF2α-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca2+]i, but caused a Y27632-sensitive constriction in α-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC20 at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca2+ sensitization and vasoconstriction independent of hydrogen peroxide.

Abstract 1707: Murine γHerpesvirus-68 Infection Induces Pulmonary Vascular Disease in S100A4 Overexpressing Mice Associated with Elastase Activity and Altered MicroRNA Expression

Human γ herpesvirus infection is linked to idiopathic pulmonary arterial hypertension (IPAH). S100A4, a metastasis gene, is highly expressed in the plexiform lesions of IPAH patients. Previously, we reported that murine γherpesvirus-68 (M1-MHV-68) infection in S100A4 overexpressing but not in C57 wild-type (WT) mice induced mild neointimal formation in pulmonary arteries (PA) associated with heightened serine elastase activity. We hypothesized that these neointimal lesions would progress during an extended reactivation phase, in the S100A4 mice in association with ongoing elastolysis and that the susceptibility in S100A4 mice was related either to viral load, inflammation or differential induction of host miRNAs in response to infection. We now report that the % of vessels with neointimal lesions progressively increased from 25 to 40%, between 3 and 6 mo after viral infection (P &lt; 0.05) in S100A mice but were not observed in WT. The extent of neointimal lesions correlated with progressive fragmentation of the elastic laminae, (P &lt; 0.001). Interestingly, these features were not sufficient to induce elevated RVSP or RVH, in the absence of muscularization or loss of distal PAs. Susceptibilty of S100A4 mice was not related to an increase in viral DNA in intra-pulmonary arteries assessed by laser capture microscopy, nor could we establish a relationship with the severity of perivascular inflammation or cytokine expression (IFN-γ and TNF-α). However, we confirmed higher lung serine elastase activity in S100A4 vs. C57 mice as early as 1 wk after viral infection. Since expression of miRNAs may be responsible for host susceptibility, we compared lungs of C57 and S100A4 mice at 6 mo using microArrays followed by qRT-PCR. Induction of miR-466 in WT mice infected with virus (P &lt; 0.01), was not observed in S100A4 mice. miR-466 can target the 3′ UTR of RUNX1, a transcription factor for serine elastase, and reduce its expression. The inability to reduce RUNX1 in S100A4 mice in response to viral infection may be related to elevated serine elastase activity. Our studies show that viral infection can lead to progressive neointimal formation and that the susceptibility of S100A4 mice associated with heightened elastase may be linked to differential expression of miR-466. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada &amp; Utah).

Regional heterogeneity of substance P-induced endothelium-dependent contraction, relaxation, and -independent contraction in rabbit pulmonary arteries

Aims The present study examined whether substance P (SP)-induced endothelium-dependent TXA 2-mediated contraction (EDC), nitric oxide (NO)-mediated relaxation (EDR), and endothelium-independent contraction (EIC) are different between the rabbit proximal and distal intrapulmonary arteries. Main methods The helically cut strips of isolated proximal and distal arteries were fixed vertically between hooks in organ bath, and changes in isometric tension were measured. Key findings SP-induced EDC was greater in the distal than proximal arteries, and EDR was greater in the proximal than distal arteries. However, under the complete blockade of NK 2 receptors and NO production, SP (10 − 9 –3 × 10 − 7 M)-induced EDC did not differ between proximal and distal arteries. Under the complete blockade of NK 2 receptors and TXA 2 production, SP (3 × 10 − 10 –3 × 10 − 8 M)-induced EDR was greater in the proximal than distal arteries. Neither contraction induced by U-46619, a TXA 2 agonist, nor relaxation by sodium nitroprusside, an NO donor, was different between both portions of the arteries. Both ionomycin (10 − 8 M)- and l-arginine (1 mM)-induced EDRs were also significantly greater in the proximal than distal arteries. Under the blockade of NK 1 receptors and NO and TXA 2 production, SP (10 − 7 M)-induced EIC was greater in the distal than proximal arteries. In summary, the capacity for NO production is higher in the proximal than distal arteries, resulting in SP-induced higher EDR and lower EDC in the proximal arteries. Significance These regional differences in responses to SP may play important roles in maintaining the homogenous distribution of blood flow in the lung.

Constriction of pulmonary artery by peroxide: role of Ca 2+ release and PKC

Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca 2+ using Fura-PE3. Ca 2+ sensitization was examined in α-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC 20 was assayed by Western blot. Peroxide (30 μM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca 2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca 2+ was affected by removal of extracellular Ca 2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCα, but had no significant effect on MYPT-1 or MLC 20 phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca 2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC 20-independent constrictor mechanism.