Stephen L Archer, M.D. - US grants

This proposal evaluates the effects of endothelium-dependent vasodilators (EDVs) on pulmonary artery (PA) endothelial cell cytosolic calcium concentration ([Ca2+]i) and nitric oxide synthesis. The importance of endothelial nitric oxide production in determining PA pressure and responsiveness to vasodilators is assessed in normal and hypertensive rats. EDRF has previously been defined by its bioassay properties. Several laboratories have proposed that EDRF bioactivity is due to the synthesis of nitric oxide from L-arginine in endothelial cells. Nitric oxide then diffuses to the adjacent vascular smooth muscle where it stimulates guanylate cyclase. It is uncertain how an EDV initiates nitric oxide synthesis; however the increase in endothelial [Ca2+]i, caused by most EDVs has been implicated as a possible signal. This "nitric oxide hypothesis" requires validation in the lung. Few investigators have correlated PA vasodilation with direct measurement of nitric oxide; most have relied on EDRF bioassays or the use of nonspecific EDRF-inhibitors. HYPOTHESIS: 1 Endothelium-dependent vasodilation in the lung is not solely the result of nitric oxide synthesis. Preliminary data showing that bradykinin (BK)- and A23187-induced PA vasodilation is associated with nitric oxide synthesis whereas that caused by acetylcholine (ACH) is not. A chemiluminescence assay will be used to determine whether the magnitude and time course of nitric oxide synthesis explains the vasodilation caused by a battery of EDVs (in normal and monocrotaline PHT rats). The specificity of putative inhibitors of nitric oxide synthesis, L-monomethyl arginine (L-NMMA) and L- nitroarginine (Nitro-Arg) will be studied using a new nitric oxide assay. 2. Nitric oxide synthesis in response to EDVs is preceded by and results from a transient rise in [Ca2+]i in PA endothelial cells. Preliminary data show that BK and A23187 increase whereas ACH reduces [Ca2+]i. The spatial and temporal characteristics of changes in [Ca2+]i which occur in response to EDVs will be measured using an imaging fluorescent microscopy system. The cytosolic "Ca2+ maps" created by this system will reveal the intracellular location and temporal dynamics of the various Ca2+ pools which account for the net change in [Ca2+]i caused by EDVs. It may be that change in a specific intracellular Ca2+ pool is responsible for EDV-induced nitric oxide synthesis. The importance of extracellular Ca2+ versus intracellular Ca2+ in increasing [Ca2+]i and initiating nitric oxide synthesis will be addressed. 3. Endothelial damage may alter the lung's capacity to generate nitric oxide and contribute to the development of pulmonary hypertension (PHT). The relationship of vascular structure to the ability of PA rings to dilate and synthesize nitric oxide will be studied in lungs from rats with monocrotaline-induced PHT.

DESCRIPTION (provided by applicant): The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated by a direct, O2-induced vasoconstrictor mechanism. Failure of functional closure can cause persistent DA. This proposal assesses a comprehensive 02 sensing pathway in human and rabbit DA, which is hypothesized to consist of a mitochondrial 02 sensor, which produces a diffusible redox mediator that regulates an effector (voltage-gated K+ channels, Kv). Preliminary data indicate that increasing PO2 hyperpolarizes mitochondrial membrane potential thus increasing H202 production and inhibiting redox-sensitive, Kv channels in DA smooth muscle cells (DASMC). The resulting membrane depolarization activates L-type Ca++ channels, promoting vasoconstriction. Kv1.5 & Kv2.1 are implicated in this mechanism because they are weakly expressed in the premature DA, which constricts weakly to 02, and their overexpression, by gene transfer, enhances DA constriction. Inhibiting certain electron transport chain (ETC) complexes mimics hypoxia: depolarizing mitochondria, decreasing H202 levels, and increasing K+ current (in DASMCs) and relaxing DA rings, consistent with a mitochondrial redox sensor. Sensor and effector pathways are examined in term human and rabbit DA and in models characterized by impaired 02 constriction and decreased Kv expression: preterm rabbit DA and ironically remodeled DA. Laser capture microdissection is used to isolate SMCs from the media of human DAs for analysis of maturational changes in DA genomic and proteomic expression profiles by quantitative RT-PCR and a ProteinChip mass spectroscopy technique. Significance: Early DA closure improves outcomes and promotes early hospital discharge. By understanding the regulation of DASMC Kv channels function/expression, one may be able to modulate the tone/patency of human DA in vivo via drugs or gene therapy. Hypotheses: 1) DA constriction results from inhibition of DASMC Kv channels by a diffusible redox messenger (H202) produced by the DASMC mitochondrial ETC. 2) Maturational changes in the DASMC expression of O2-sensitive Kv channels or -subunits underlies the reduced responses of preterm rabbit DAs to 02 and contributes to their high incidence of patency. 3) Augmenting expression of O2-sensitive, DASMC Kv channels in preterm or ionically remodeled DA (using an SMC-specific adenovirus) will enhance 02 constriction.

DESCRIPTION (provided by applicant): This proposal responds to the broad challenge area (04): Clinical Research and specific challenge topic 04-HL-102: Develop Integrative Strategies to Elucidate the Mechanisms of Lung Diseases. Pulmonary arterial hypertension (PAH) is a syndrome characterized by obstructive vascular remodeling, inflammation and vasoconstriction of small pulmonary arteries. Despite recent therapeutic advances, 1-year mortality rates remains high (~15%). Although abnormalities of the platelets, endothelium and adventitia contribute critically to the pathogenesis of PAH, excessive proliferation of pulmonary arterial smooth muscle cells (PASMC) is a major contributor to the obstructive vascular pathology. This challenge proposal explores 2 newly-recognized abnormalities that promote PASMC proliferation. We recently discovered that the mitochondrial network is disrupted in PAH PASMC and noted that this is related to the proliferative diathesis of these cells. Fragmentation of the mitochondrial network appears to reflect impaired mitochondrial fusion and is associated with 2 related abnormalities: 1) normoxic activation of the master hypoxic transcription factor, hypoxia inducible factor (HIF-1a) and 2) downregulation of mitofusin-2. Normally, mitochondria rapidly join and break apart through highly regulated processes called fusion and fission, respectively. The balance of fusion and fission dynamically regulates the integrity of the reticulum. Fusion is regulated by SNARE-like proteins called mitofusin-1 and mitofusin-2. Fusion redistributes mitochondrial proteins/genes, protecting the cell from oxidant stress, apoptosis and mitochondrial DNA mutations. Impaired fusion alters mitochondrial membrane potential, impairs respiration and promotes SMC proliferation. We evaluate the hypothesis that a HIF-1a-mediated mitofusin-2 deficiency promotes PASMC proliferation and contributes to PAH. Relevant to PAH, mitofusin-2 is a brake on SMC proliferation. Indeed, when first cloned, mitofusin-2 was named hyperplasia suppressor gene. Mitofusin-2 gene therapy reduces intimal hyperplasia in a systemic arterial injury model. HIF-1a is known to downregulate mitofusin-2 expression. Impaired fusion and normoxic HIF-1a activation are found in humans with PAH and fawn-hooded rats (FHR), a strain that spontaneously develops PAH. PUBLIC HEALTH RELEVANCE: We are investigating the mechanism of pulmonary arterial hypertension (PAH) in Fawn Hooded Rats (FHR), and have identified problems in the FHR's mitochondria, namely lower production of hydrogen peroxide and fragmentation of the mitochondrial network, which create a pseudohypoxic environment that favors rapid cell growth and blood vessel blockage. Preliminary studies show that FHR suffer from activation of an hypoxia inducible factor" (HIF-1a) and a related break-up of the mitochondrial network due to a deficiency of mitofusin 2 (which triggers excessive growth of arterial smooth muscle cells and blocks the lung circulation). This proposal seeks to understand the role of the HIF-1a and mitofusin in PAH and develop therapies (inhibition of HIF-1 a and supplementation of mitofusin-2) to restore mitochondrial form and function and cure PAH.

DESCRIPTION (provided by applicant): The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated within minutes by O2-induced vasoconstriction. Functional closure (vasoconstriction) stops right to left shunting of blood and promotes anatomical closure. Failure of these processes leads to persistent ductus arteriosus, a common form of congenital heart disease in premature infants. Although endothelial-derived mediators modulate DA tone, O2 exerts a direct constrictor effect. During the first 5 years of this grant we showed that the DA's O2-sensing pathway consists of a sensor (the mitochondrial electron transport chain), which produces a diffusible mediator (H2O2), that inhibits voltage-gated K+ channels, such as Kv1.5. At birth, O2-induced increases in mitochondrial H2O2 in DA smooth muscle cells (DASMC) promote constriction by several mechanisms: Kv channel inhibition, direct activation of O2-sensitive calcium channels and rho kinase activation. Moreover, preterm DASMC are relatively deficient in these mechanisms, explaining the prevalence of persistent DA in preterm DA. This renewal focuses on a discovery made during a search for splice variants of Kv1.5 in human DASMC. We found a novel K+ channel, Human Oxygen-Sensitive K+ channel (HOSK), that when heterologously expressed creates a current that is voltage-gated, displays K+ specificity (Rb>K>>Cs>Na), and is 4- aminopyridine sensitive. HOSK appears to contribute to the resting membrane potential in human DASMC. HOSK siRNA reduces the O2-sensitive current in human DASMC. HOSK cDNA corresponds to a 3.0 kb neuronal, expressed sequence tag (EST) and has an unusual coding mechanism. HOSK and collagen 12(I) have identical mRNA with the much smaller 21 kDa HOSK resulting from initiation of translation at an internal ribosomal entry site (IRES). In silico modeling suggests that HOSK may have four hydrophobic domains (HD), a unique K+ selectivity filter (GVL, rather than the typical GYG amino acid sequence) and a variant voltage sensor. Phylogenetic analysis suggests HOSK originated in amniotes. In this proposal, the relative importance of HOSK versus canonical O2-sensitive voltage-gated K+ channels, Kv1.5 and Kv2.1, will be compared in term human DA, and two models of impaired O2 constriction: preterm rabbit DA and ionically remodeled human DA. Hypothesis 1: HOSK is a novel K+ channel, arising independent of the canonical K+ channel family. Hypothesis 2: HOSK contributes to DA constriction and augmenting HOSK expression will enhance O2- constriction in preterm rabbit DA and ionically remodeled human DA. Significance: The proposed experiments will contribute to our understanding of the normal mechanism of DA constriction and functional closure of the human DA. HOSK, hidden by its complex encoding mechanism and unique structure may offer a new explanation for how O2 causes functional closure and shed light on the link between DA constriction and fibrous obliteration of the DA. PUBLIC HEALTH RELEVANCE: The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. The DA closes at birth by a by an O2-induced, vasoconstrictor mechanism that is intrinsic to the smooth muscle cells. Failure of functional closure (vasoconstriction) can cause persistent DA. During the first 5 years of this grant we showed that the DA's O2-sensing pathway consists of a mitochondrial sensor, which produces diffusible H2O2, that inhibits voltage-gated K+ channels, such as Kv1.5. This renewal assesses a novel voltage-gated K+ channel, Human Oxygen-Sensitive K+ channel (HOSK). Hidden by its complex encoding mechanism within the collagen a2(1) gene HOSK has a unique structure and explain how O2 causes functional closure of the DA and extend our basic knowledge of K+ channel structure/function.

DESCRIPTION (provided by applicant): The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated within minutes by O2-induced vasoconstriction. Functional closure (vasoconstriction) stops right to left shunting of blood and promotes anatomical closure. Failure of these processes leads to persistent ductus arteriosus, a common form of congenital heart disease in premature infants. During the first 5 years of this grant we showed that the DA's O2- sensing pathway consists of a sensor (the mitochondrial electron transport chain), which produces a diffusible mediator (H2O2), that inhibits voltage-gated K+ channels, such as Kv1.5. At birth, O2-induced increases in mitochondrial H2O2 in DA smooth muscle cells (DASMC) promote constriction by several mechanisms: Kv channel inhibition, direct activation of O2-sensitive calcium channels and rho kinase activation. Moreover, preterm DASMC are relatively deficient in these mechanisms, explaining the prevalence of persistent DA in preterm DA. This renewal focuses on a discovery made during a search for splice variants of Kv1.5 in human DASMC. We found a novel K+ channel, Human Oxygen-Sensitive K+ channel (HOSK), that when heterologously expressed creates a current that is voltage-gated, displays K+ specificity (Rb>K>>Cs>Na), and is 4- aminopyridine sensitive. HOSK appears to contribute to the resting membrane potential in human DASMC. HOSK siRNA reduces the O2-sensitive current in human DASMC. HOSK cDNA corresponds to a 3.0 kb neuronal, expressed sequence tag (EST) and has an unusual coding mechanism. HOSK and collagen 12(I) have identical mRNA with the much smaller 21 kDa HOSK resulting from initiation of translation at an internal ribosomal entry site (IRES). In silico modeling suggests that HOSK may have four hydrophobic domains (HD), a unique K+ selectivity filter (GVL, rather than the typical GYG amino acid sequence) and a variant voltage sensor. Phylogenetic analysis suggests HOSK originated in amniotes. In this proposal, the relative importance of HOSK versus canonical O2-sensitive voltage-gated K+ channels, Kv1.5 and Kv2.1, will be compared in term human DA, and two models of impaired O2 constriction: preterm rabbit DA and ionically remodeled human DA. Aim 1: HOSK is a novel K+ channel, arising independent of the canonical K+ channel family. Aim 2: HOSK contributes to DA constriction and augmenting HOSK expression will enhance O2- constriction in preterm rabbit DA and ionically remodeled human DA. Aim 3: HIF-11 shifts translation of the collagen gene away from HOSK toward collagen 21(1). HOSK, hidden by its complex encoding mechanism and unique structure, may offer a new explanation for how O2 causes constriction and will shed light on the link between constriction and fibrous obliteration of the DA. Since HOSK may have arisen independent of the canonical K+ channel family, exploring its structure-function relationship will extend our fundamental knowledge K+ channels.

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by obstruction of the pulmonary vasculature due (in part) to idiopathic hyper-proliferation of pulmonary arterial smooth muscle cells (PASMC). Preliminary data suggest that this proliferative diathesis relates to excessive mitochondrial fission caused by activation of dynamin related protein 1 (DRP-1). In this proposal we will define the molecular basis for dysregulation of fission in human PAH and explore the nature of the link between a mitochondrial fission-fusion cycle and the cell cycle. We also evaluate whether the requirement of these hyper-proliferative cells for high rates of mitochondrial fission presents an Achilles hee that can be therapeutically targeted. Experiments are performed in human PAH PASMC and lungs and in experimental models (induced by monocrotaline or SU5416 + hypoxia). Preliminary data indicate that human PAH PASMC have fragmented mitochondria, largely due to increased fission. We have developed metrics that use mitochondrial-targeted, photoactivated, green fluorescent protein (mito-paGFP) and 2-photon confocal microscopy to quantify mitochondrial fission. Increased fission in PAH appears to result from phosphorylation of DRP-1 at Serine 616 by the key mitotic regulatory kinase, Cyclin B1- CDK1, thus linking fission and mitosis. Elevated cytosolic calcium in PAH also promotes DRP-1-mediated fission by: a) activation of calmodulin kinase and b) calcineurin-mediated dephosphorylation of an inhibitory form of DRP- 1 (Phos-Ser637). Thus, increased activity of calcineurin, cyclin B1-CDK1 and DRP-1 promote fission and proliferation in human PAH PASMC; conversely, DRP-1 inhibition (via the small molecule inhibitor, mdivi-1, or siDRP-1) decreases PASMC proliferation and, in the case of mdivi-1, regresses PAH in vivo. Hypothesis: Pathologic DRP-1 activation increases fission and promotes excessive PASMC proliferation in PAH. Corollary: DRP-1 inhibition prevents cell cycle progression, causing G2-M phase arrest. Anti-fission therapies may constitute an antiproliferative therapy for PAH. Aim 1) Are DRP-1 activation and fission required for the hyperproliferation of PASMC in human PAH? Aim 2) Does post-translational DRP-1 modification regulate PASMC proliferation in PAH? Aim 3) Does inhibition of mitochondrial fission have therapeutic benefit in experimental PAH? Innovation and Impact: This proposal is innovative in recognizing that mitochondrial checkpoints, related to DRP-1 activation and fission, regulate cell cycle progression. The concept that mitochondrial fission is tightly linked to cell cycle progression via shared regulatory kinases is also novel. The discovery that inhibiting DRP-1 (directly or by targeting its regulatory kinases) is anti-proliferative offers a new, anti-fission profusion, therapeutic paradigm for PAH. The translational potential of this proposal is enhanced by careful correlation of results in rodent PAH models with findings in human PAH lungs/cells from a well-characterized cohort. This is the first study to exploit PAH's reliance on rapid fission to devise novel anti-fission, anti-proliferative therapies for PAH.