Macritchie, Neil Alexander
Modifying factors in pulmonary arterial hypertension.
PhD thesis, University of Glasgow.
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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.
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