Background: Vascular remodeling is a pathological process central to many cardiovascular diseases (CVDs). Vascular injury promotes a complex interplay between distinct cell populations in the vessel wall that results in endothelial dysfunction and local inflammation, which in turn stimulate vascular smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) deposition, leading to neointimal thickening and stenosis. Different signaling pathways, including the melanocortin system (MCS), modulate vascular inflammation and oxidative stress. The MCS refers to a set of hormonal and paracrine signaling pathways that include five G protein-coupled receptors (GPCRs), peptide agonists derived from the post-translational processing of the proopiomelanocortin (POMC) prohormone, ancillary proteins, and endogenous antagonists. This system regulates a remarkably diverse array of physiological functions and host reactions. Preclinical investigations indicate that activation of certain melanocortin receptor (MCR) subtypes, primarily MC1R, could be a novel strategy to control inflammatory disorders. A local MCS has been described in endothelial cells (EC) of the cutaneous and cerebral microcirculation. The melanocortin alpha-melanocyte stimulating hormone (α-MSH) has been shown to modulate blood vessel tone by enhancing nitric oxide (NO)-cyclic-guanosine mono-phosphate dependent relaxation responses through endothelial MC1R. Besides, treatment with MCR agonists was able to prevent the development of vascular dysfunction and attenuate plaque inflammation in pre-established atherosclerosis. We sought to investigate whether human macrovascular ECs and SMCs express locally any MCR and what function MCRs exert. Methods: Expression of MCS components in primary human aortic (HAoEC), coronary artery ECs (HCAEC) and human aortic smooth muscle cells (HAoSMC) were assessed by real-time PCR (qRT-PCR), western blot (WB), immunocytochemistry (ICC) and immunohistochemistry (IHC). Intracellular cyclic adenosine-mono-phosphate (cAMP) concentrations were measured by an ELISA assay. Intracellular EC calcium (Ca2+) concentrations were analyzed using a fluorescent Ca2+ indicator. Gap closure assays were used for assessing in vitro cell migration of ECs and SMCs. SMC transmigration was assessed by a transwell migration assays along a chemoattractant gradient (chemotaxis), and proliferation by incorporation and measurement of a nucleoside analog in newly synthesized DNA. Genome-wide gene expression profiling was performed in migrating HAoECs and HAoSMCs after stimulation with α-MSH in time-course experiments, using microarrays and RNA sequencing, respectively. SMC phenotypic changes and associated biomarkers were visualized and measured by ICC and qPCR. Results: We showed that primary human aortic ECs and SMCs express functionally active MCRs. ECs express MC1R, but not other MCR subtypes nor the precursor hormone POMC. MC1R engagement with α-MSH, its highest-affinity natural ligand, accelerates cell migration in an in vitro directional migration assay. This was associated with enhancing in Ca2+ signaling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in ECs undergoing directional migration assay allowed identifying dynamic co-regulation of genes involved in the extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing: all these pathways have a well-established influence on EC motility. SMCs express MC1R and MC4R, but not POMC. In vitro stimulation with α-MSH promoted human aortic SMC differentiation from a synthetic to a contractile phenotype with a spindle-shape conformation. α-MSH promoted significant human aortic SMC elongation with peak effects at 6-12 h after treatment. Stimulation with α-MSH enhanced gene and protein expression of α-Smooth-Muscle Actin (α-SMA) and Smooth-Muscle-22-α (SM22α), which are markers of the contractile phenotype. MCR activation by α-MSH slowed down migration both in the gap-closure assay (peak effect at 9-12 h) and in the transwell migration assay. We confirmed that MC1R is involved in this process because its inhibition with a specific antagonist (MSG-606) restored the normal migration rate. Furthermore, MCR activation slightly decreased HAoSMC proliferation. We then investigated which pathways were activated downstream the receptor in migrating SMC, by analyzing the transcriptome and specific phosphoproteins. We found a negative modulation of the p38 mitogen-activated protein kinase (p38MAPK)/heat shock factor 1 (HSF1) pathway along with a significant reduction in p38-MAPK phosphorylation, which likely is the mechanism of action underlying MC1R-related modulation of SMC migration. Conclusions: These results provide evidence of a novel function of peripherally expressed MCRs, i.e. regulation of EC and SMC motility, ultimately contributing to vessel homeostasis. In addition, this is the first study that seeks to uncover the possible influences of MCRs expressed in macrovascular arteries on the pathophysiology of vascular remodeling. Indeed, results from this study suggest novel, potentially promising therapeutic targets for prevention and healing of vascular remodeling: targeting the MCRs may result in controlling vessel inflammation, healing endothelial dysfunction, and inhibiting SMC phenotypic switching.

LOCALLY EXPRESSED MELANOCORTIN RECEPTORS MODULATE HUMAN MACROVASCULAR REMODELING / V. Alfieri ; CO-TUTOR: G. COLOMBO, F. SAPORITI ; COORDINATOR: A. CATAPANO. - : . DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Dec 17. ((31. ciclo, Anno Accademico 2018. [10.13130/alfieri-valentina_phd2018-12-17].

LOCALLY EXPRESSED MELANOCORTIN RECEPTORS MODULATE HUMAN MACROVASCULAR REMODELING

V. Alfieri
2018-12-17

Abstract

Background: Vascular remodeling is a pathological process central to many cardiovascular diseases (CVDs). Vascular injury promotes a complex interplay between distinct cell populations in the vessel wall that results in endothelial dysfunction and local inflammation, which in turn stimulate vascular smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) deposition, leading to neointimal thickening and stenosis. Different signaling pathways, including the melanocortin system (MCS), modulate vascular inflammation and oxidative stress. The MCS refers to a set of hormonal and paracrine signaling pathways that include five G protein-coupled receptors (GPCRs), peptide agonists derived from the post-translational processing of the proopiomelanocortin (POMC) prohormone, ancillary proteins, and endogenous antagonists. This system regulates a remarkably diverse array of physiological functions and host reactions. Preclinical investigations indicate that activation of certain melanocortin receptor (MCR) subtypes, primarily MC1R, could be a novel strategy to control inflammatory disorders. A local MCS has been described in endothelial cells (EC) of the cutaneous and cerebral microcirculation. The melanocortin alpha-melanocyte stimulating hormone (α-MSH) has been shown to modulate blood vessel tone by enhancing nitric oxide (NO)-cyclic-guanosine mono-phosphate dependent relaxation responses through endothelial MC1R. Besides, treatment with MCR agonists was able to prevent the development of vascular dysfunction and attenuate plaque inflammation in pre-established atherosclerosis. We sought to investigate whether human macrovascular ECs and SMCs express locally any MCR and what function MCRs exert. Methods: Expression of MCS components in primary human aortic (HAoEC), coronary artery ECs (HCAEC) and human aortic smooth muscle cells (HAoSMC) were assessed by real-time PCR (qRT-PCR), western blot (WB), immunocytochemistry (ICC) and immunohistochemistry (IHC). Intracellular cyclic adenosine-mono-phosphate (cAMP) concentrations were measured by an ELISA assay. Intracellular EC calcium (Ca2+) concentrations were analyzed using a fluorescent Ca2+ indicator. Gap closure assays were used for assessing in vitro cell migration of ECs and SMCs. SMC transmigration was assessed by a transwell migration assays along a chemoattractant gradient (chemotaxis), and proliferation by incorporation and measurement of a nucleoside analog in newly synthesized DNA. Genome-wide gene expression profiling was performed in migrating HAoECs and HAoSMCs after stimulation with α-MSH in time-course experiments, using microarrays and RNA sequencing, respectively. SMC phenotypic changes and associated biomarkers were visualized and measured by ICC and qPCR. Results: We showed that primary human aortic ECs and SMCs express functionally active MCRs. ECs express MC1R, but not other MCR subtypes nor the precursor hormone POMC. MC1R engagement with α-MSH, its highest-affinity natural ligand, accelerates cell migration in an in vitro directional migration assay. This was associated with enhancing in Ca2+ signaling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in ECs undergoing directional migration assay allowed identifying dynamic co-regulation of genes involved in the extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing: all these pathways have a well-established influence on EC motility. SMCs express MC1R and MC4R, but not POMC. In vitro stimulation with α-MSH promoted human aortic SMC differentiation from a synthetic to a contractile phenotype with a spindle-shape conformation. α-MSH promoted significant human aortic SMC elongation with peak effects at 6-12 h after treatment. Stimulation with α-MSH enhanced gene and protein expression of α-Smooth-Muscle Actin (α-SMA) and Smooth-Muscle-22-α (SM22α), which are markers of the contractile phenotype. MCR activation by α-MSH slowed down migration both in the gap-closure assay (peak effect at 9-12 h) and in the transwell migration assay. We confirmed that MC1R is involved in this process because its inhibition with a specific antagonist (MSG-606) restored the normal migration rate. Furthermore, MCR activation slightly decreased HAoSMC proliferation. We then investigated which pathways were activated downstream the receptor in migrating SMC, by analyzing the transcriptome and specific phosphoproteins. We found a negative modulation of the p38 mitogen-activated protein kinase (p38MAPK)/heat shock factor 1 (HSF1) pathway along with a significant reduction in p38-MAPK phosphorylation, which likely is the mechanism of action underlying MC1R-related modulation of SMC migration. Conclusions: These results provide evidence of a novel function of peripherally expressed MCRs, i.e. regulation of EC and SMC motility, ultimately contributing to vessel homeostasis. In addition, this is the first study that seeks to uncover the possible influences of MCRs expressed in macrovascular arteries on the pathophysiology of vascular remodeling. Indeed, results from this study suggest novel, potentially promising therapeutic targets for prevention and healing of vascular remodeling: targeting the MCRs may result in controlling vessel inflammation, healing endothelial dysfunction, and inhibiting SMC phenotypic switching.
CAMERA, MARINA
CATAPANO, ALBERICO LUIGI
Settore BIO/14 - Farmacologia
LOCALLY EXPRESSED MELANOCORTIN RECEPTORS MODULATE HUMAN MACROVASCULAR REMODELING / V. Alfieri ; CO-TUTOR: G. COLOMBO, F. SAPORITI ; COORDINATOR: A. CATAPANO. - : . DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2018 Dec 17. ((31. ciclo, Anno Accademico 2018. [10.13130/alfieri-valentina_phd2018-12-17].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/605171
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