Since pulmonary hypertension (PH) develops in a relatively small proportion of patients with a genetic abnormality, e.g., a mutation in bone morphogenetic protein receptor (BMPR)2, or in conjunction with an environmental exposure, e.g., HIV or amphetamine (AMPH) abuse, aberrant genes and environmental factors are likely additive or synergistic in causing disease. Our preliminary studies with cultured cells and transgenic mice with dysfunctional BMPR2 suggest that common processes are amplified when the aberrant gene interacts with an adverse environmental factor. For example, we determined that loss of BMPR2 causes abnormal assembly of elastin fibers by pulmonary arterial (PA) smooth muscle cells (SMC) and fibroblasts (Fibro), and we hypothesize that accelerated degradation of these fibers would occur in response to the inflammation resulting from a viral infection. Similarly, loss of BMPR2 reduces cell survival signals in PA endothelial cells (EC), and hypoxia and AMPH further decrease the same signal, resulting in amplification of DNA damage and PA EC apoptosis. The goal of the present proposal is therefore to investigate how loss of BMPR2 signaling causes impaired assembly of elastin fibers and also renders EC susceptible to apoptosis and DNA damage, and whether, in combination with infection or AMPH, the consequences and amplification of these abnormalities increases severity or impairs resolution of PH. In preliminary studies we showed that loss of BMPR2 in PA SMC and Fibro impairs elastin fiber assembly by reducing fibrillin proteins and activating transforming growth inhibitory factor (TGIF), to prevent synthesis of tropoelastin.
In Specific Aim 1, we will determine whether loss of BMPR2 decreases fibrillin-stabilizing proteins such as syndecans and ADAMTS disintegrins. We will establish whether the elevated level of p-p38 observed with reduced BMPR2 activates TGIF, and whether EGF phosphorylation is a necessary intermediary. We will assess the contribution of PA EC to formation of elastin fibers by SMC, and whether this is compromised by loss of BMPR2. In Bmpr2+/-Alk3+/- mice, or in mice with deleted Bmpr2 in SMC or Fibro, we will elucidate whether elastase activity, produced by perivascular inflammatory cells in response to a vasculotropic virus, will cause the abnormal PA elastin fibers to degrade, promoting adverse remodeling, including neointimal formation, and severe PH. In preliminary studies we showed a progressive loss of pAkt with reduced BMPR2, hypoxia and AMPH. Thus, in Specific Aim 2 we will investigate whether this is related to a similar phosphatase activated by reduced BMPR2, hypoxia and AMPH, and whether progressive perturbation in pAkt function causes DNA damage and EC apoptosis by impairing p53 dependent gene regulation. We will determine, in Bmpr2+/-Alk3+/- and in mice with loss of Bmpr2 in EC, whether AMPH mediated amplification of DNA damage worsens hypoxic PH and vascular changes and prevents reversal. Understanding the contribution of BMPR2 dysfunction to environmental insults will facilitate approaches to prevent and minimize PH in patients at risk.
Our proposal addresses why only a small proportion of patients with a genetic abnormality, e.g., a mutation in bone morphogenetic protein receptor (BMPR)2, or with environmental exposure, e.g., HIV-like virus or amphetamine use, develop pulmonary arterial hypertension (PAH). With the goal to better understand the PAH risk and how to minimize it, we study cultured vascular cells, including those from patients with PAH, and mice where BMPR2 is deleted in specific vascular cells. We investigate the common features of the two pathways related to disease, one where loss of BMPR2 coupled to infection and inflammation potentiates the degradation of elastic fibers and the consequent obliterative pulmonary arterial remodeling, and the second where amphetamine causes damage of DNA and death of endothelial cells.
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