We aimed to investigate the role of endothelin-mediated vasoconstriction following acute pulmonary thromboembolism (APTE). Thirteen anesthetized piglets (~25 kg) were ventilated with 0 PEEP. Cardiac output (Qt) and wedge pressure (Pw) were measured by a Swan Ganz catheter, along with arterial and venous blood gases. APTE was induced by autologous blood clots (~0.8 g/kg, 12-16 pieces) via a jugular venous catheter at time = 0 minutes until the mean pulmonary arterial pressure (Ppa) was about 2.5 times the baseline at 30 minutes. Eight control animals (Group 1) received only normal saline afterward, while the remaining five (Group 2) received at time = 40-minute saline plus Tezosentan, a nonspecific endothelin antagonist. The drug was initially given as an intravenous bolus (10 mg/kg), followed by an infusion (2 mg/min) until the end of the experiment at 2 hours. Hemodynamic data were measured before APTE and then at 30-minute intervals. Pulmonary vascular resistance index (PVRI) was calculated as (Ppa-Pw)/CI, where CI was cardiac index or Qt/W (body weight). Fluorescent microspheres (FMS) were used to mark regional blood flows and ventilation for cluster analysis. PVRI acutely increased within minutes and remained high despite some recovery over time. With Tezosentan treatment, the results showed that endothelin-mediated vasoconstriction persisted significantly up to 2 hours and accounted for about 25% of the increase in PVRI while clot obstruction accounted for the remaining 75%. CI remained relatively constant throughout. Tezosentan also affected PVRI indirectly by mitigating the shift of regional blood flow back to the embolized areas over time, possibly by attenuating vasoconstriction in the nonembolized areas. We conclude that following APTE, although the increased PVRI is mostly due to mechanical embolic obstruction, secondary factors such as vasoconstriction and pattern of regional blood flow over time also play important roles.

Endotoxemia impairs hypoxic pulmonary vasoconstriction which leads to systemic hypoxemia. This derogation is attributable to increased activity of nitric oxide synthase 2 and arginase metabolism. Gene expression analysis has shown increased expression of ornithine decarboxylase in lungs of endotoxemic mice, a downstream enzyme of arginase metabolism. The aim of this study was to investigate whether inhibition of ornithine decarboxylase increases hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice. Mice received lipopolysaccharides or saline intraperitoneal, and hypoxic pulmonary vasoconstriction was measured using an isolated perfused mouse lung model. Additional mice with and without endotoxemia were pretreated with the ornithine decarboxylase-inhibitor difluoromethylornithine before examination of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was defined as the difference of pulmonary arterial pressure between normoxic and hypoxic ventilation. In addition, lung tissue was analyzed using real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry. Lipopolysaccharides caused an up-regulation of ornithine decarboxylase mRNA level (2.73 ± 0.19-fold increase, p < 0.05) as well as ornithine decarboxylase protein level (4.05 ± 0.37-fold increase, p < 0.05). Endotoxemia attenuated hypoxic pulmonary vasoconstriction when compared with untreated control mice (26.3 ± 9.7% vs. 67.0 ± 17.5%). Difluoromethylornithine (20, 100, 500 mg kg-1 body weight intraperitoneal) restored hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice in a dose-dependent way (25.8 ± 9.9%, 57.3 ± 17.2%, 62.3 ± 12.4%) and decreased hypoxic pulmonary vasoconstriction in control mice (53.6 ± 13.6%, 40.0 ± 14.9%, 35.9 ± 12.4%). These results show that endotoxemia induces ornithine decarboxylase expression and suggest that ornithine decarboxylase contributes to the endotoxemia-induced impairment of hypoxic pulmonary vasoconstriction. Inhibition of ornithine decarboxylase might be a target in the therapy of diseases with inflammation impaired hypoxic pulmonary vasoconstriction, like the sepsis-associated acute respiratory distress syndrome (ARDS).

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O2 saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca2+ signaling and Ca2+ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca2+ or Ca2+ influx through various Ca2+-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca2+ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca2+ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca2+-sensing receptors (by 30 µM NPS2143, an allosteric Ca2+-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca2+ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca2+ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca2+-mediated activation of Ca2+-sensing receptors and the cell-cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is a compensatory physiological mechanism in the lung that optimizes the matching of ventilation to perfusion and thereby maximizes gas exchange. Historically, HPV has been primarily studied in isolated perfused/ventilated lungs; however, the results of these studies have varied greatly due to different experimental conditions and species. Therefore, in the present study, we utilized the mouse isolated perfused/ventilated lung model for investigation of the role of extracellular Ca(2+) and caveolin-1 and endothelial nitric oxide synthase expression on HPV. We also compared HPV using different perfusate solutions: Physiological salt solution (PSS) with albumin, Ficoll, rat blood, fetal bovine serum (FBS), or Dulbecco's Modified Eagle Medium (DMEM). After stabilization of the pulmonary arterial pressure (PAP), hypoxic (1% O2) and normoxic (21% O2) gases were applied via a ventilator in five-minute intervals to measure HPV. The addition of albumin or Ficoll with PSS did not induce persistent and strong HPV with or without a pretone agent. DMEM with the inclusion of FBS in the perfusate induced strong HPV in the first hypoxic challenge, but the HPV was neither persistent nor repetitive. PSS with rat blood only induced a small increase in HPV amplitude. Persistent and repetitive HPV occurred with PSS with 20% FBS as perfusate. HPV was significantly decreased by the removal of extracellular Ca(2+) along with addition of 1 mM EGTA to chelate residual Ca(2+) and voltage-dependent Ca(2+) channel blocker (nifedipine 1 μM). PAP was also reactive to contractile stimulation by high K(+) depolarization and U46619 (a stable analogue of thromboxane A2). In summary, optimal conditions for measuring HPV were established in the isolated perfused/ventilated mouse lung. Using this method, we further confirmed that HPV is dependent on Ca(2+) influx.

This study aimed to identify receptors mediating sphingosine-1-phosphate (S1P)-induced vasoconstriction in the normotensive and chronic hypoxia-induced hypertensive rat pulmonary circulation. In isolated perfused lungs from normoxic rats, infusion of S1P caused a sustained vasoconstriction, which was not reduced by combinational pretreatment with the dual S1P(1 and 3) receptor antagonist VPC23019 and the S1P(2) receptor antagonist JTE013. The S1P(4) receptor agonists phytosphingosine-1-phospate and VPC23153, but not the dual S1P(1 and 3) receptor agonist VPC24191, caused dose-dependent vasoconstrictions. In hypertensive lungs from chronically hypoxic rats, the vasoconstrictor responses to S1P and VPC23153 were markedly enhanced. The S1P(4) receptor agonist VPC 23153 caused contraction of isolated pulmonary but not of renal or mesenteric arteries from chronically hypoxic rats. S1P(4) receptor protein as well as mRNA were detected in both normotensive and hypertensive pulmonary arteries. In contrast to what has been reported in the systemic circulation and mouse lung, our findings raise the possibility that S1P(4) receptor plays a significant role in S1P-induced vasoconstriction in the normotensive and hypertensive rat pulmonary circulation.

Enhanced vasoconstriction is increasingly identified as an important contributor to the development of pulmonary hypertension. Chronic hypoxia results in enhanced Rho kinase mediated Ca2+ sensitization contributing to pressure-dependent pulmonary arterial tone as well as augmented vasoconstriction to endothelin-1 and depolarizing stimuli. We sought to investigate the interaction between these vasoconstrictor stimuli in isolated, pressurized, pulmonary arteries. We used the K+ ionophore, valinomycin, to clamp membrane potential (Vm) to investigate the role of membrane depolarization in endothelin-1 and pressure-dependent constriction, and endothelin-1 receptor inhibitors to determine whether membrane depolarization or stretch signal through endothelin-1 receptors. Clamping Vm prevented pressure-dependent tone, but not enhanced vasoconstriction to endothelin-1 following chronic hypoxia. Furthermore, endothelin-1 receptor inhibition had no effect on either pressure-dependent tone or vasoconstriction to KCl. As Src kinases contribute to both pressure-dependent tone and enhanced endothelin-1 vasoconstriction following chronic hypoxia, we further investigated their role in depolarization-induced vasoconstriction. Inhibition of Src kinases attenuated enhanced vasoconstriction to KCl. We conclude that membrane depolarization contributes to pressure-dependent tone but not enhanced vasoconstriction to ET-1, and that Src kinases serve as upstream mediators facilitating enhanced Rho kinase-dependent vasoconstriction following chronic hypoxia.

Acute pulmonary embolism is the third most common cause of cardiovascular death. Pulmonary embolism increases right ventricular afterload, which causes right ventricular failure, circulatory collapse and death. Most treatments focus on removal of the mechanical obstruction caused by the embolism, but pulmonary vasoconstriction is a significant contributor to the increased right ventricular afterload and is often left untreated. Pulmonary thromboembolism causes mechanical obstruction of the pulmonary vasculature coupled with a complex interaction between humoral factors from the activated platelets, endothelial effects, reflexes and hypoxia to cause pulmonary vasoconstriction that worsens right ventricular afterload. Vasoconstrictors include serotonin, thromboxane, prostaglandins and endothelins, counterbalanced by vasodilators such as nitric oxide and prostacyclins. Exogenous administration of pulmonary vasodilators in acute pulmonary embolism seems attractive but all come with a risk of systemic vasodilation or worsening of pulmonary ventilation-perfusion mismatch. In animal models of acute pulmonary embolism, modulators of the nitric oxide-cyclic guanosine monophosphate-protein kinase G pathway, endothelin pathway and prostaglandin pathway have been investigated. But only a small number of clinical case reports and prospective clinical trials exist. The aim of this review is to give an overview of the causes of pulmonary embolism-induced pulmonary vasoconstriction and of experimental and human investigations of pulmonary vasodilation in acute pulmonary embolism.

Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 μM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.

Pharmacogenomics is the study of how genetic variations influence the response to drugs, by correlating gene expression with the drug's efficacy and toxicity. This concept has recently been successfully applied in oncology. To test its applicability to PAH, we examined two experimental models of the disease: mice with deletion of the Vasoactive Intestinal Peptide gene (VIP(- /-)); and rats injected with monocrotaline (MCT). Since the two models express comparable phenotypic features, we analyzed their particular gene alterations, with special reference to genes related to pulmonary vasoconstriction, vascular remodeling, and inflammation. We then compared the phenotypic and genotypic responses in each model to treatment with the same drug, VIP. In untreated VIP(-/-) mice there was over-expression of almost all genes promoting vasoconstriction/ proliferation, as well as inflammation, and under-expression of all vasodilator/anti-proliferative genes. As expected, treatment with VIP fully corrected both the key PAH features, and all gene expression alterations. MCT-treated rats showed two distinct sets of alterations. One, similar to that in VIP(- /-) mice, i.e., tended to promote vascular remodeling and inflammation, e.g., up-regulation of myosin polypeptides, procollagen, and some inflammatory genes. The other was a set of opposite alterations that suggested an effort to modulate the PAH, e.g., up-regulation of the VIP and NOS3 genes. In this model, VIP treatment failed to correct many of the genotypic abnormalities, and, in parallel, incompletely corrected the phenotypic changes as well. This preliminary proof-of-concept study demonstrates the importance of genomic information in determining therapeutic outcome, and thus in selecting personalized therapy. Full validation of the merits of pharmacogenomics must await studies of lungs from patients with different forms of PAH.

Pulmonary vasoconstriction and vascular remodeling are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with idiopathic pulmonary arterial hypertension (IPAH). An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation, which causes pulmonary vascular remodeling. Store-operated Ca(2+) entry (SOCE), induced by depletion of stored Ca(2+) in the sarcoplasmic reticulum (SR), can increase [Ca(2+)](cyt) in PASMC independent of other means of Ca(2+) entry. Stromal interaction molecule (STIM) proteins, STIM1 and STIM2, were recently identified as both sensors for store depletion and signaling molecules to open store-operated Ca(2+) channels. We previously reported that SOCE was significantly enhanced in PASMC from IPAH patients compared to PASMC from normotensive control subjects. Enhanced SOCE plays an important role in the pathophysiological changes in PASMC associated with pulmonary arterial hypertension. In this study, we examine whether the expression level of STIM1 and STIM2 is altered in IPAH-PASMC compared to control PASMC and whether these putative changes in STIM1/2 expression level are responsible for enhanced SOCE and proliferation in IPAH-PASMC. Compared to control PASMC, the protein expression level of STIM2 was significantly increased whereas STIM1 protein expression was not significantly changed. In IPAH-PASMC, siRNA-mediated knockdown of STIM2 decreased SOCE and proliferation, while knockdown of STIM2 in control PASMC had no effect on either SOCE or proliferation. Overexpression of STIM2 in control PASMC failed to enhance SOCE or proliferation. These data indicate that enhanced protein expression of STIM2 is necessary, but not sufficient, for enhanced SOCE and proliferation of IPAH-PASMC.

Pulmonary hypertension is a chronic vascular disease characterized by pulmonary vasoconstriction and pulmonary arterial remodeling. Pulmonary arterial remodeling is mainly due to small pulmonary arterial wall thickening and lumen occlusion. Previous studies have described intravascular changes in lung sections using histopathology, but few were able to obtain a fine detailed image of the pulmonary vascular system. In this study, we used Microfil compounds to cast the pulmonary arteries in a rat model of monocrotaline-induced pulmonary hypertension. High-quality images that enabled quantification of distal pulmonary arterial branching based on the number of vessel bifurcations/junctions were demonstrated in this model. The branch and junction counts of distal pulmonary arteries significantly decreased in the monocrotaline group compared to the control group, and this effect was inversely proportional to the mean pulmonary artery pressure observed in each group. The patterns of pulmonary vasculature and the methods for pulmonary vessel casting are presented to provide a basis for future studies of pulmonary arterial remodeling due to pulmonary hypertension and other lung diseases that involve the remodeling of vasculature.

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

Pulmonary arterial (PA) wall modifications are key pathological features of pulmonary hypertension (PH). Although such abnormalities correlate with heightened phosphorylation of c-Jun N-terminal kinases 1/2 (JNK1/2) in a rat model of PH, the contribution of specific JNK isoforms to the pathophysiology of PH is unknown. Hence, we hypothesized that activation of either one, or both JNK isoforms regulates PA remodeling in PH. We detected increased JNK1/2 phosphorylation in the thickened vessels of PH patients' lungs compared to that in lungs of healthy individuals. JNK1/2 phosphorylation paralleled a marked reduction in MAP kinase phosphatase 1 (JNK dephosphorylator) expression in patients' lungs. Association of JNK1/2 activation with vascular modification was confirmed in the calf model of severe hypoxia-induced PH. To ascertain the role of each JNK isoform in pathophysiology of PH, wild-type (WT), JNK1 null (JNK1-/-), and JNK2 null (JNK2-/-) mice were exposed to chronic hypoxia (10% O2 for six weeks) to develop PH. In hypoxic WT lungs, an increase in JNK1/2 phosphorylation was associated with PH-like pathology. Hallmarks of PH pathophysiology, i.e. excessive accumulation of extracellular matrix and vessel muscularization with medial wall thickening, was also detected in hypoxic JNK1-/- lungs, but not in hypoxia-exposed JNK2-/- lungs. However, hypoxia-induced increases in right ventricular systolic pressure (RVSP) and in right ventricular hypertrophy (RVH) were similar in all three genotypes. Our findings suggest that JNK2 participates in PA remodeling (but likely not in vasoconstriction) in murine hypoxic PH and that modulating JNK2 actions might quell vascular abnormalities and limit the course of PH.

While it is well established that the haemodynamic cause of hypoxic pulmonary hypertension is increased pulmonary vascular resistance, the molecular pathogenesis of the increased resistance remains incompletely understood. Macrophage migration inhibitory factor is a pleiotropic cytokine with endogenous tautomerase enzymatic activity as well as both intracellular and extracellular signalling functions. In several diseases, macrophage migration inhibitory factor has pro-inflammatory roles that are dependent upon signalling through the cell surface receptors CD74, CXCR2 and CXCR4. Macrophage migration inhibitory factor expression is increased in animal models of hypoxic pulmonary hypertension and macrophage migration inhibitory factor tautomerase inhibitors, which block some of the functions of macrophage migration inhibitory factor, and have been shown to attenuate hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. However, because of the multiple pathways through which it acts, the integrated actions of macrophage migration inhibitory factor during the development of hypoxic pulmonary hypertension were unclear. We report here that isolated lungs from adult macrophage migration inhibitory factor knockout (MIF-/- ) mice maintained in normoxic conditions showed greater acute hypoxic vasoconstriction than the lungs of wild type mice (MIF+/+ ). Following exposure to hypoxia for three weeks, isolated lungs from MIF-/- mice had significantly higher pulmonary vascular resistance than those from MIF+/+ mice. The major mechanism underlying the greater increase in pulmonary vascular resistance in the hypoxic MIF-/- mice was reduction of the pulmonary vascular bed due to an impairment of the normal hypoxia-induced expansion of the alveolar capillary network. Taken together, these results demonstrate that macrophage migration inhibitory factor plays a central role in the development of the pulmonary vascular responses to chronic alveolar hypoxia.