VEGF and Sympathetic Perivascular Nerves Contribute to Hypoxic Remodeling of Ovine Cranial Arteries

Chronic hypoxia complicates many pregnancies and can result in postnatal pathologies that include compromised fetal cardiovascular structure and function. Mechanisms involved remain unclear. Because hypoxia increases production of VEGF, known to modulate smooth muscle (SM) phenotype, this thesis explored the hypothesis that VEGF contributes to hypoxic fetal vascular remodeling through direct effects on SM cells and indirectly through perivascular nerves. Using a chronic hypoxia sheep model, this work demonstrated that: 1) hypoxia potently upregulates VEGF receptor expression but not endogenous VEGF level in fetal ovine carotid arteries; 2) both chronic hypoxia and VEGF exert similar effects on smooth muscle contractile proteins; 3) both chronic hypoxia and VEGF exert similar effects on contractile protein colocalizations; and lastly, sympathetic autonomic nerves contribute to hypoxic reorganization of structure and function of vascular contractile proteins. Together, these findings advance understanding of how hypoxia precipitates fetal vascular remodeling and offer an essential first step toward finding new treatments for infants that survive in-utero hypoxia

Role of Protein Kinase G in Regulating Vascular Tone in Hypoxic Ovine Cerebral Arteries

Nitric oxide (NO) and its precursors have been used clinically to induce vasorelaxation long before the specific mechanism of how this operates was understood. With the discovery in 1977 of the influence of NO on guanylate cyclase, and the subsequent elucidation of the NO pathway, the role of cyclic guanidine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to be the main mediator of NO-induced vasorelaxation. Less well understood is the behavior of this pathway in conditions of long term hypoxia (LTH). It was later shown that LTH attenuates the ability of PKG to mediate vasorelaxation. How this operates is the focus of this investigation. Here we tested the hypothesis that LTH attenuates NO-induced vasorelaxation by reducing the ability of PKG to phosphorylate its target proteins that mediate vascular relaxation and contraction. A prominent target of PKG involved with vasorelaxation is the BK channel alpha protein. To examine the influence of LTH on PKG-induced vasorelaxation, and to demonstrate the relative influence of the BK channel in these conditions, we used carotid and middle cerebral arteries from normoxic and chronically hypoxic fetal and adult sheep. These arteries were denuded of endothelium and used in concentration-response relations using 5-HT as a contractant and measured contractile force. To measure the relative influence of the BK channel in these arteries, we used the selective BK channel blocker iberiotoxin. From this we demonstrated that while hypoxia affects both BK and non-BK channel mechanisms, the influence of LTH on the BK channel is dominant in the loss of PKG-mediated vasorelaxation. We also examined whether hypoxia attenuates PKG expression and specific activity, as well as the BK channel alpha and beta proteins. Using Western blotting and enzyme kinase activity assays, we learned that LTH has a modest influence on BK channel proteins expression, and no influence on PKG expression or activity. We next examined whether LTH influences protein organization, which could potentially control PKG-mediated vasorelaxation in normoxic and hypoxic conditions. Using confocal microscopy, we learned that LTH strongly alters protein organization, and specifically the proximal association of three proteins associated with vasorelaxation, namely PKG, BK-alpha and BK-beta

Modifying factors in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a debilitating disease of small pulmonary resistance arteries with vasoconstriction and vascular remodelling contributing to the disease pathology. A genetic basis for the disease was linked to heterozygous loss of function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene. The mutation is found in the majority of familial PAH cases and a significant number of apparently sporadic cases. The low penetrance of the disease in families carrying BMPR2 mutations and the absence of mutations in the majority of idiopathic patients indicates that BMPR2 deficiency alone is insufficient to induce PAH. It is generally accepted PAH has a multi-factorial pathology with endogenous and environmental factors acting in concert with genetic pre-disposition to create the disease phenotype. Enhancement of the serotonin (5-HT) system has been implicated in PAH with the 5-HT transporter (5-HTT) receiving the most attention as a modifying gene in the development of PAH and there is compelling animal and human data implicating a role for increased expression of the 5-HTT as a modulating factor. The aim of this study was to investigate if genetic pre-disposition interacts with other additional modifying factors to create the symptoms of PAH. Transgenic mice overexpressing the 5-HTT (5-HTT+), deficient in BMPR2 (BMPR2+/-) or a double transgenic (5-HTT+/BMPR2+/-) were employed in addition to mice lacking tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme for the synthesis of 5-HT, and therefore lacking peripheral 5-HT (Tph1-/-). Additional known or suspected modifying factors assessed in these genetic models were hypoxia, dexfenfluramine (Dfen) and its major metabolite nordexfenfluramine (NDfen), 5-HT, bone morphogenetic protein-2 (BMP-2), KCNQ channels and the role of gender. Mice were examined in vivo for evidence of a pulmonary hypertensive phenotype following exposure to hypoxia and Dfen. Female 5-HTT+ mice were the only group tohave a rise in two indices of PAH - namely right ventricular pressure (RVP) and vascular remodelling - in room air. Female 5-HTT+ mice also had an exaggerated pulmonary hypertensive phenotype in hypoxia. BMPR2+/- mice, were, unexpectedly least susceptible to hypoxic induced increases in RVP although female mice deficient in BMPR2 (both BMPR2+/- and 5-HTT+/BMPR2+/-) had more extensive vascular remodelling under hypoxia compared with WT and 5-HTT+ mice. Male mice did not express the phenotypic changes just outlined. No synergistic effect was observed between 5-HTT+ and BMPR2+/- that resulted in a more pronounced pulmonary hypertensive phenotype. WT and BMPR2+/- mice were chronically oral-dosed with Dfen. Female mice from both genotypes developed similar degrees of PAH. Male mice did not develop elevated RVP but BMPR2+/- males did have evidence of vascular remodelling, although at a lower level than the females. Female Tph1-/- mice did not develop PAH following Dfen indicating Dfen associated PAH is dependent on peripheral 5-HT synthesis. The presence of intact 5-HT synthesis was also associated with an increased vasoconstrictor response to 5-HT in isolated intralobar pulmonary arteries (IPAs), a situation not paralleled with the other serotonergic vasoconstrictors, Dfen and NDfen, indicating differing mechanisms of action underlying the respective vasoconstrictor responses. The vasoconstrictor action of 5-HT, Dfen, NDfen and the KCNQ potassium channel blocker linopirdine were all assessed in IPAs. Pulmonary arteries from BMPR2+/- mice showed enhanced vasoconstriction to 5-HT and NDfen. 5-HTT+ and 5-HTT+/BMPR2+/- mice showed enhanced vasoconstriction to NDfen but decreased vasoconstriction to 5-HT. Female 5-HTT+/BMPR2+/- mice were the only group tested to show significantly greater vasoconstriction to Dfen compared with WT. The vasoconstrictor response to linopirdine was significantly reduced in BMPR2+/- mice but neither linopirdine nor BMP-2 affected 5- HT induced vasoconstriction. Female gender is an established risk factor for PAH. To investigate possible events that may underlie this risk, male (testosterone) and female (estradiol and 2-methoxyestradiol (2-ME)) sex hormones were assessed for their vasoactive properties in IPAs. All three hormones relaxed pre-constricted vessels but only at supraphysiological (>0.1 µM) concentrations. Each hormone also reduced the vasoconstriction exerted by 5-HT at 10-5 M in male mice but not in females. No such effect, however, was observed in either gender at a physiological (10-9 M) concentration. NDfen induced vasoconstriction was also unaffected by 10-9 M estradiol. Finally, male and female mouse lungs were assessed for protein expression of 5-HT and BMPR2 signalling compounds (p-Smad1/5/8, p-ERK1/2 and p-p38 MAPK). Female mouse lungs displayed higher expression of the mitogenic mediator p-ERK1/2 than male mouse lungs with the other proteins unchanged. In conclusion, this study confirms overexpression of the 5-HTT as a trigger for elevated RVP and vascular remodelling in mice and a cause of more severe hypoxic PAH. BMPR2+/- mice are phenotypically normal in room air and show divergent pulmonary effects following hypoxia with loss of BMPR2 seemingly attenuating hypoxic induced increases in RVP but causing a simultaneous worsening of vascular remodelling, this latter effect consistent with the important role BMPR2 has in maintaining vascular integrity. Dfen induced PAH in mice was found to be dependent on peripheral 5-HT synthesis with BMPR2 mutation not acting as a risk factor. Loss of BMPR2 can enhance vasoconstriction to serotonergic agonists and when combined with overexpression of the 5-HTT, leads to a dramatic increase in sensitivity to Dfen induced vasoconstriction. Evidence was also found for altered KCNQ channel function in transgenic animals. Unexpectedly, female gender emerged as the most crucial risk factor for PAH in this thesis

Hypoxia-induced responses of porcine pulmonary veins.

The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research

Effects of hypoxia on the human lung

A large number of animal studies have investigated the effects of hypoxia on the pulmonary vasculature and hypoxic pulmonary vasoconstriction (HPV) is now established as an important homeostatic mechanism for perfusion-ventilation matching in the lung. However, there is a conspicuous lack of studies investigating HPV in human pulmonary vascular preparations. In comparison to the pulmonary vasculature only a limited number of studies have previously investigated the effects of acute hypoxia on the airways. In this thesis the effects of hypoxia on the human pulmonary vasculature and airways was investigated in a number of ex vivo human lung models.In isolated human pulmonary arteries and veins and in ex vivo perfused and ventilated human lungs it was found that hypoxia caused a vasodilation. This finding is at variance with a large body of published literature and it is suggested that a neuronal mechanism could play an important role in the regulation of pulmonary vascular tone under hypoxic conditions in vivo which is not present in the ex vivo models used in this thesis.In exploring the effects of hypoxia on the pulmonary vasculature it was identified that the pulmonary veins could play a more significant role in the regulation of pulmonary vascular resistance than was previously believed. It was also found that exposure of the pulmonary veins to hypoxia precipitated a phasic activity which was suggestive of an automaticity and that the isolated human pulmonary vein model could therefore be used to investigate arrhythmic activity and the efficacy of new antiarrhythmic agents. Exposure of ex vivo perfused and ventilated human lungs to hypoxia caused a reduction in the rate of oedema formation and it is hypothesised that this effect could be mediated by an inhibition of pulmonary vascular endothelial cell contraction and an increase in endothelial barrier function.In isolated human bronchi and ex vivo human lungs hypoxia caused a robust and reversible bronchodilation. The mechanism of hypoxic bronchodilation (HBD) was investigated and it was found that HBD could be mediated by a reversal of the calcium sensitisation mechanism. This mechanism represents a significant therapeutic target for the future development of effective bronchodilator therapies which are not subject to the limitations of GPCR agonists (desensitisation).Studies investigating the effects of changing oxygen concentrations on bronchial tone identified that oxygen concentrations above ambient levels (21%) caused a robust contraction of human airways which could have significant implications for the clinical use of oxygen therapy in constrictive airway disease.In investigating the putative role of hydrogen sulphide (H₂S) as an oxygen sensor it was found that H₂S is an effective vasodilator and bronchodilator in the human lung and could have significant therapeutic potential in the treatment of human pulmonary disease.Further studies have been planned to build on the significant findings in this thesis

The role of connexin 43 in the function of systemic and pulmonary blood vessels

Background: Connexins are membrane channel-forming proteins that play a pivotal role in direct intercellular communication within the vasculature. They are crucial in mediating various aspects of vascular physiology such as vasodilation and vasoconstriction, and also play a significant role in vascular pathology. The aim of this research is to elucidate the roles of connexins, particularly connexin 43 (Cx43), in the regulation of systemic and pulmonary circulations. The hypothesis is that Cx43 critically influences vascular reactivity and remodelling, which is particularly evident in pathological conditions like pulmonary hypertension. By investigating these roles, this study seeks to deepen our understanding of Cx43’s multifaceted functions in both physiological and pathophysiological contexts, including its involvement in disorders such as atherosclerosis, systemic hypertension, and pulmonary hypertension (PH). Introduction: Connexin 43 (Cx43) is the most abundant isoform within the vasculature, and this thesis will examine its role in regulation of both the systemic and pulmonary circulation. In chapter 3 and 4, the role of Cx43 will be investigated in the systemic and pulmonary circulations respectively using mouse thoracic aortae, mouse intra-lobar pulmonary arteries (IPAs) and lungs. Pulmonary hypertension is a severe condition characterised by both pulmonary vascular remodelling and abnormal vasoconstriction, resulting in increased pulmonary vascular resistance and eventually leading to right-sided heart failure and death. Given the significant contribution of pulmonary artery fibroblasts (PAFs) to pulmonary vascular remodelling, chapter 5 will focus on examining the role of connexin 43 in proliferation and migration of mouse PAFs (MPAFs). Methods: Thoracic aortae from 4- to 6-month-old mice and lungs and IPAs from 2- to 3-month-old mice were dissected free from age-matched female and male wild-type (WT) mice and connexin 43 heterozygous (Cx43+/−) mice from a C57BL/6 background. Using pentobarbital sodium (60mg/kg) and lidocaine (4mg/kg), mice were euthanized by intraperitoneal injection (i.p.). In all experiments, the role of Cx43 in the systemic and pulmonary vasculature was investigated using both pharmacological and genetic approaches. The gap junction blocker 37,43Gap27 was employed to pharmacologically inhibit Cx43 function. Additionally, Cx43+/− mice were used, wherein Cx43 expression is genetically reduced, not completely inhibited, providing a model to understand the impact of lower Cx43 levels on vascular function. The use of Cx43−/− mice, which completely lack Cx43, was not feasible due to lethal developmental defects that these mice experience, making them unsuitable for such studies. In the first part of this study, wire myography was used to assess both systemic and pulmonary vascular reactivity of thoracic aortae and IPAs. NO production was then investigated in whole thoracic aortae and lung tissues using a Sievers 280 analyser. Western blot was also used to investigate the protein expression of Cx43 and eNOS in the thoracic aortae and lungs. Cell culture was then used to examine the role of Cx43 in MPAFs. MPAFs were explanted from main and branch pulmonary arteries and were exposed to normoxic or hypoxic (5% O2) conditions for 24h. Proliferation and migration were assessed using an automated cell counter or a scratch assay respectively. Results: Pharmacological inhibition and genetic reduction of Cx43 had effects on vascular reactivity and NO production in both the systemic and pulmonary circulations. In all of the experiments presented in this thesis on systemic and pulmonary vasoreactivity and NO assays, no significant differences between sexes were found. This study found that Cx43 associates with endothelial nitric oxide synthase and exists in the same protein complexes within whole thoracic aortae and lungs from both genotypes. This study further revealed that Cx43 significantly promotes the proliferation and migration of MPAFs under hypoxic conditions. This could ultimately lead to the remodelling of the pulmonary vasculature and subsequently leading to pulmonary hypertension. Conclusion: This study has shown that Cx43 plays a role in systemic and pulmonary vasoreactivity and interacts with NO signalling pathways, indicating its potential as a pharmacological target. It is further demonstrated in this study that Cx43 is involved in hypoxic-induced cell proliferation and migration. Thus, Cx43 may be involved in the pulmonary vascular remodelling in response to hypoxia. This cellular process is of significant interest in the context of pulmonary vascular changes, and these findings should be seen as a foundation for further understanding the complex role of Cx43 in conditions like PH. Future research should investigate deeper into elucidating the specific molecular pathways involved and assessing the implications of targeting Cx43 therapeutically

Effect of hypoxia on the cardiovascular sphingolipid system

Sphingosine kinase 1 (SK1) catalyses the synthesis of the important bioactive sphingolipid sphingosine-1-phosphate (S1P), that has an important role in vascular tone regulation and cardioprotection against ischaemia/reperfusion injury. The work presented in this thesis describes the influence of short periods of hypoxia on expression of SK1 in vascular endothelium and how this may regulate vascular function. The aims were achieved by using wire myography to study vascular function and confocal microscopy for the studies of expression and distribution of SK1 under normoxic and hypoxic conditions. In the first study, it was found that exposure of isolated rat coronary artery to a short period of hypoxia increases SK1 expression and ser225 phosphorylation. It was also demonstrated that the hypoxia-induced increase in SK1 expression was reduced by pre-treatment with cycloheximide, a protein synthesis inhibitor, SKi, a non-selective SK inhibitor and PF543, a selective SK1 inhibitor. However, pre-treatment with proteasomal and/or lysosomal inhibitors did not increase SK1 expression under normoxia or hypoxia. Similarity, SK1 expression was also increased in aortic endothelium following exposure to short-term hypoxia and this effect was also inhibited by cycloheximide, SKi and PF543. Collectively, these data suggest that hypoxia increases SK1 synthesis in coronary and aortic endothelium. Moreover, the SKi-induced reduction in SK1 expression in coronary endothelium was reversed by proteasomal and/or lysosomal inhibitors, indicating that SKi stimulates both proteasomal and lysosomal degradation of SK1 under normoxia and hypoxia. In chapter two, it was demonstrated that S1P and CYM5541, an S1P3 agonist, induced dose-dependent relaxation in endothelium-intact aortic rings, whereas the S1P1 agonist SEW2871 was without effect. The S1P stimulated relaxation was significantly enhanced in endothelium-intact aortic rings subjected to short-term hypoxia and this effect was entirely endothelium-dependent. Interestingly, the vasorelaxation response to S1P was inhibited by pre-treatment with SKi and PF543 but not ROMe, a selective SK2 inhibitor under both normoxia and hypoxia. A nitric oxide synthase inhibitor also inhibited the S1P-induced relaxation in aortic rings. Moreover, the enhanced relaxation response to S1P due to hypoxia was maintained in aortae obtained from spontaneously hypertensive Wistar Kyoto rats. These findings suggest that the vasorelaxation response to S1P under normoxia and the enhanced response under hypoxia are mediated by SK1 and NO. In chapter five, it was found that hypoxia did not change the SK1b expression in HUVECs and pre-treatment with SKi or cycloheximide exerted no effect under both normoxia and hypoxia. However, proteasomal and/or lysosomal inhibitors increased SK1 expression under hypoxic conditions. In heart tissue, no significant difference was seen in expression of SK1 following exposure to hypoxia. However, SK1 expression was reduced by pre-treatment with SK inhibitors and cycloheximide under normoxia but not hypoxia. SK1a was identified in heart tissue, which is more sensitive to the degradation-induced by SK inhibitors than SK1b. In summary, the results of this study imply that short-term hypoxia induces an increase in SK1 expression in coronary and aortic vascular endothelium. The increased SK1 induced by hypoxia appears to mediate the enhanced vasorelaxation response to S1P in endothelium-intact aortae. In HUVECs and heart tissue, it is likely that hypoxia induces resistance of SK1 to SK inhibitor-induced downregulation through a compensatory increase in SK1 expression