using-vivo-tissue-cell-explant-approaches-to-study-morphogenesis

Using <em>In Vivo</em> and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Yale University School of Medicine

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.

Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites.

The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [(32)P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2 in vitro reduces the V(max) of immunopurified eNOS by a factor of 2 but has no effect on the K(A) for CaM or calcium. Additionally, [(32)P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM ("phosphonull" S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the "phosphomimetic" Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using Escherichia coli-expressed and phenyl-Sepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis.

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1beta and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation.

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza.

Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure.

Radial construction of an arterial wall.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling associated with obliteration of pulmonary arterioles and formation of plexiform lesions composed of hyperproliferative endothelial and vascular smooth-muscle cells. Here we describe a microRNA (miRNA)-dependent association between apelin (APLN) and fibroblast growth factor 2 (FGF2) signaling in pulmonary artery endothelial cells (PAECs). APLN deficiency in these cells led to increased expression of FGF2 and its receptor FGFR1 as a consequence of decreased expression of miR-424 and miR-503, which directly target FGF2 and FGFR1. miR-424 and miR-503 were downregulated in PAH, exerted antiproliferative effects in PAECs and inhibited the capacity of PAEC-conditioned medium to induce the proliferation of pulmonary artery smooth-muscle cells. Reconstitution of miR-424 and miR-503 in vivo ameliorated pulmonary hypertension in experimental models. These studies identify an APLN-dependent miRNA-FGF signaling axis needed for the maintenance of pulmonary vascular homeostasis.

An endothelial apelin-FGF link mediated by miR-424 and miR-503 is disrupted in pulmonary arterial hypertension.

Arteries consist of an inner single layer of endothelial cells surrounded by layers of smooth muscle and an outer adventitia. The majority of vascular developmental studies focus on the construction of endothelial networks through the process of angiogenesis. Although many devastating vascular diseases involve abnormalities in components of the smooth muscle and adventitia (i.e., the vascular wall), the morphogenesis of these layers has received relatively less attention. Here, we briefly review key elements underlying endothelial layer formation and then focus on vascular wall development, specifically on smooth muscle cell origins and differentiation, patterning of the vascular wall, and the role of extracellular matrix and adventitial progenitor cells. Finally, we discuss select human diseases characterized by marked vascular wall abnormalities. We propose that continuing to apply approaches from developmental biology to the study of vascular disease will stimulate important advancements in elucidating disease mechanism and devising novel therapeutic strategies.

Development and pathologies of the arterial wall.

Excess smooth muscle accumulation is a key component of many vascular disorders, including atherosclerosis, restenosis, and pulmonary artery hypertension, but the underlying cell biological processes are not well defined. In pulmonary artery hypertension, reduced pulmonary artery compliance is a strong independent predictor of mortality, and pathological distal arteriole muscularization contributes to this reduced compliance. We recently demonstrated that embryonic pulmonary artery wall morphogenesis consists of discrete developmentally regulated steps. In contrast, poor understanding of distal arteriole muscularization in pulmonary artery hypertension severely limits existing therapies that aim to dilate the pulmonary vasculature but have modest clinical benefit and do not prevent hypermuscularization. Here, we show that most pathological distal arteriole smooth muscle cells, but not alveolar myofibroblasts, derive from pre-existing smooth muscle. Furthermore, the program of distal arteriole muscularization encompasses smooth muscle cell dedifferentiation, distal migration, proliferation, and then redifferentiation, thereby recapitulating many facets of arterial wall development.

Recapitulation of developing artery muscularization in pulmonary hypertension.

Vascular biology: Brain vessels squeezed to death.

Excess and ectopic smooth muscle cells (SMCs) are central to cardiovascular disease pathogenesis, but underlying mechanisms are poorly defined. For instance, pulmonary hypertension (PH) or elevated pulmonary artery blood pressure is a devastating disease with distal extension of smooth muscle to normally unmuscularized pulmonary arterioles. We identify novel SMC progenitors that are located at the pulmonary arteriole muscular-unmuscular border and express both SMC markers and the undifferentiated mesenchyme marker platelet-derived growth factor receptor-β (PDGFR-β). We term these cells "primed" because in hypoxia-induced PH, they express the pluripotency factor Kruppel-like factor 4 (KLF4), and in each arteriole, one of them migrates distally, dedifferentiates, and clonally expands, giving rise to the distal SMCs. Furthermore, hypoxia-induced expression of the ligand PDGF-B regulates primed cell KLF4 expression, and enhanced PDGF-B and KLF4 levels are required for distal arteriole muscularization and PH. Finally, in PH patients, KLF4 is markedly up-regulated in pulmonary arteriole smooth muscle, especially in proliferating SMCs. In sum, we have identified a pool of SMC progenitors that are critical for the pathogenesis of PH, and perhaps other vascular disorders, and therapeutic strategies targeting this cell type promise to have profound implications.

Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension.

The aorta is the largest artery in the body, yet processes underlying aortic pathology are poorly understood. The arterial media consists of circumferential layers of elastic lamellae and smooth muscle cells (SMCs), and many arterial diseases are characterized by defective lamellae and excess SMCs; however, a mechanism linking these pathological features is lacking. In this study, we use lineage and genetic analysis, pharmacological inhibition, explant cultures, and induced pluripotent stem cells (iPSCs) to investigate supravalvular aortic stenosis (SVAS) patients and/or elastin mutant mice that model SVAS. These experiments demonstrate that multiple preexisting SMCs give rise to excess aortic SMCs in elastin mutants, and these SMCs are hyperproliferative and dedifferentiated. In addition, SVAS iPSC-derived SMCs and the aortic media of elastin mutant mice and SVAS patients have enhanced integrin β3 levels, activation, and downstream signaling, resulting in SMC misalignment and hyperproliferation. Reduced β3 gene dosage in elastin-null mice mitigates pathological aortic muscularization, SMC misorientation, and lumen loss and extends survival, which is unprecedented. Finally, pharmacological β3 inhibition in elastin mutant mice and explants attenuates aortic hypermuscularization and stenosis. Thus, integrin β3-mediated signaling in SMCs links elastin deficiency and pathological stenosis, and inhibiting this pathway is an attractive therapeutic strategy for SVAS.

Integrin β3 inhibition is a therapeutic strategy for supravalvular aortic stenosis.

Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term.

mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice.

Article	Year
Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. Biochemistry\| PubMed ID: 12501214	2002
Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 14736917	2004
KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. The Journal of experimental medicine\| PubMed ID: 15136591	2004
Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza. Science translational medicine\| PubMed ID: 20375003	2010
Radial construction of an arterial wall. Developmental cell\| PubMed ID: 22975322	2012
An endothelial apelin-FGF link mediated by miR-424 and miR-503 is disrupted in pulmonary arterial hypertension. Nature medicine\| PubMed ID: 23263626	2013
Development and pathologies of the arterial wall. Cellular and molecular life sciences : CMLS\| PubMed ID: 24071897	2014
Recapitulation of developing artery muscularization in pulmonary hypertension. Cell reports\| PubMed ID: 24582963	2014
Vascular biology: Brain vessels squeezed to death. Nature\| PubMed ID: 24670635	2014
Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension. Science translational medicine\| PubMed ID: 26446956	2015
Integrin β3 inhibition is a therapeutic strategy for supravalvular aortic stenosis. The Journal of experimental medicine\| PubMed ID: 26858344	2016
mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 28751568	2017
Basic research: Where do new endothelial cells come from in the injured heart? Nature reviews. Cardiology\| PubMed ID: 28770866	2017

Daniel M. Greif

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