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Clinical and translational research has played a major role in advancing our understanding of pulmonary hypertension (PH), including pulmonary arterial hypertension and other forms of PH with severe vascular remodelling ( e.g. chronic thromboembolic PH and pulmonary veno-occlusive disease). However, PH remains an incurable condition with a high mortality rate, underscoring the need for a better transfer of novel scientific knowledge into healthcare interventions. Herein, we review recent findings in pathology (with the questioning of the strict morphological categorisation of various forms of PH into pre- or post-capillary involvement of pulmonary vessels) and cellular mechanisms contributing to the onset and progression of pulmonary vascular remodelling associated with various forms of PH. We also discuss ways to improve management and to support and optimise drug development in this research field., Competing Interests: Conflict of interest: M. Humbert reports personal fees from Actelion and Merck, and grants and personal fees from Bayer and GSK, outside the submitted work. Conflict of interest: C. Guignabert has nothing to disclose. Conflict of interest: S. Bonnet has nothing to disclose. Conflict of interest: P. Dorfmüller reports personal fees from MSD, Bayer, Actelion and Roche, outside the submitted work. Conflict of interest: J.R. Klinger reports grants for animal study of extra cellular vesicles from United Therapeutics, outside the submitted work. Conflict of interest: M.R. Nicolls has nothing to disclose. Conflict of interest: A.J. Olschewski has nothing to disclose. Conflict of interest: S.S. Pullamsetti has nothing to disclose. Conflict of interest: R.T. Schermuly has nothing to disclose. Conflict of interest: K.R. Stenmark reports grants from ContraFect, personal fees for advisory board work from Pfizer, New York, personal fees for membership of a steering Committee Member (Entelligence Awards Program) from Actelion, and personal fees for scientific advisory board work from Janssen Research and Development, outside the submitted work. Conflict of interest: M. Rabinovitch reports use of FK506 for the treatment of PAH. The Board of Trustees of Leland Stanford Junior University, assignee. Patent PCT/US2012035793., (Copyright ©ERS 2019.)

Rationale: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension., Objective: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity., Methods and Results: We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis., Conclusions: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.

Objective: Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome., Approach and Results: Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs., Conclusions: Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome., (© 2014 American Heart Association, Inc.)

Knowledge of the pathobiology of pulmonary hypertension (PH) continues to accelerate. However, fundamental gaps remain in our understanding of the underlying pathological changes in pulmonary arteries and veins in the different forms of this syndrome. Although PH primarily affects the arteries, venous disease is increasingly recognized as an important entity. Moreover, prognosis in PH is determined largely by the status of the right ventricle, rather than the levels of pulmonary artery pressures. It is increasingly clear that although vasospasm plays a role, PH is an obstructive lung panvasculopathy. Disordered metabolism and mitochondrial structure, inflammation, and dysregulation of growth factors lead to a proliferative, apoptosis-resistant state. These abnormalities may be acquired, genetically mediated as a result of mutations in bone morphogenetic protein receptor-2 or activin-like kinase-1, or epigenetically inherited (as a result of epigenetic silencing of genes such as superoxide dismutase-2). There is a pressing need to better understand how the pathobiology leads to severe disease in some patients versus mild PH in others. Recent recognition of a potential role of acquired abnormalities of mitochondrial metabolism in the right ventricular myocytes and pulmonary vascular cells suggests new therapeutic approaches, diagnostic modalities, and biomarkers. Finally, dissection of the role of pulmonary inflammation in the initiation and promotion of PH has revealed a complex yet fascinating interplay with pulmonary vascular remodeling, promising to lead to novel therapeutics and diagnostics. Emerging concepts are also relevant to the pathobiology of PH, including a role for bone marrow and circulating progenitor cells and microribonucleic acids. Continued interest in the interface of the genetic basis of PH and cellular and molecular pathogenetic links should further expand our understanding of the disease., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Rationale: S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products)., Objective: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension., Methods and Results: Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells., Conclusions: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

Background: We previously reported that administration of elastase inhibitors reverses fatal pulmonary arterial hypertension (PAH) in rats by inducing smooth muscle cell (SMC) apoptosis. We showed in pulmonary artery (PA) organ culture that the mechanism by which elastase inhibitors induce SMC apoptosis involves repression of matrix metalloproteinase (MMP) activity and subsequent signaling through alphavbeta3-integrins and epidermal growth factor receptors (EGFRs). This suggests that blockade of these downstream effectors may also induce regression of PAH., Methods and Results: In this study, we first showed in PA organ culture that MMP inhibition or alphavbeta3-integrin blockade with agents in clinical and preclinical use (SC-080 and cilengitide, respectively) mediates SMC apoptosis and regression of medial hypertrophy. We also documented similar results with an EGFR tyrosine kinase inhibitor. We then induced PAH in rats by injection of monocrotaline and, at day 21, began a 2-week treatment with SC-080, cilengitide, or the EGFR inhibitor PKI166. No vehicle- or cilengitide-treated animal survived beyond 2 weeks. Administration of SC-080 resulted in 44% survival at 2 weeks, and PKI166 therapy resulted in 78% and 54% survival in daily or 3-times-weekly treated animals, respectively. Four weeks after cessation of PKI166, we documented survivals of 50% and 23% in the 2 treatment groups, associated with reductions in pulmonary pressure, right ventricular hypertrophy, and abnormally muscularized distal arteries., Conclusions: We propose that selective blockade of EGFR signaling may be a novel strategy to reverse progressive, fatal PAH.

We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

Objective: Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease., Methods and Results: Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation., Conclusions: Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.

Postcardiac transplant coronary arteriopathy is associated with tumor necrosis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth muscle cell (SMC) migration into the subendothelium, resulting in occlusive neointimal formation. Because expression of inducible nitric oxide synthase (iNOS) is elevated in neointimal formation after transplantation and upregulated in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induction of fibronectin synthesis in coronary artery (CA) SMCs is mediated by nitric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxygen and nitrogen intermediates in CA SMCs (P<0.05). This correlated with increased NO production (P<0.05) and fibronectin synthesis (P<0.05). TNF-alpha induction of fibronectin synthesis was abrogated by the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (P<0.05) or the flavonoid-containing enzyme inhibitor diphenyleneiodonium (DPI) (P<0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P<0.05). Northern blotting showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. TNF-alpha increased expression of light chain 3 (LC-3), a protein shown previously to facilitate fibronectin mRNA translation through its interaction with an adenosine-uracil rich element (ARE) in the 3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays using CA SMC lysates revealed protein binding complexes with radiolabeled oligonucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effect inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whereby cytokines regulate mRNA translation of extracellular matrix proteins through NO-dependent modulation of RNA binding protein interaction with mRNA.

We cloned a rat vascular chymase (RVCH) from smooth muscle cells (SMCs) that converts angiotensin I to II and is up-regulated in SMC from spontaneously hypertensive vs. normotensive rats. To determine whether increased activity of RVCH is sufficient to cause hypertension, transgenic mice were generated with targeted conditional expression of RVCH to SMC, with the use of the tetracycline-controlled transactivator (tTA). We confirmed conditional expression of RVCH by mRNA, protein, and chymase activity in the absence, but not in the presence, of dietary doxycycline. The systolic blood pressure (mmHg), measured by carotid artery cannulation at 10-12 weeks of age, was higher in tTA+/RVCH+ mice than in nonbinary transgenic littermates (136 +/- 4 vs. 109 +/- 3) (P < 0.05), as were the diastolic and mean pressures. Hypertension was completely reversed by doxycycline, suggesting a causal link with chymase expression. Medial thickening of mesenteric arteries from tTA+/RVCH+ mice vs. littermates (0.82 +/- 0.1 vs. 0.42 +/- 0.02) (P < 0.05) was associated with increased SMC proliferation, as judged by positive immunoreactivity, with the use of an antibody to the proliferating cell nuclear antigen. These structural changes were prevented by doxycycline. Perfusion myography of mesenteric arteries from tTA+/RVCH+ mice also revealed increased vasoconstriction in response to phenylephrine and impaired metacholine-induced vasodilatation when compared with littermate controls or with the doxycyline-treated group. Our studies suggest that up-regulation of this vascular chymase is sufficient to cause a hypertensive arteriopathy, and that RVCH may be a candidate gene and a therapeutic target in patients with high blood pressure.

While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell-expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.

Elastases degrade the extracellular matrix, releasing growth factors and chemotactic peptides, inducing glycoproteins such as tenascin, and thereby promoting vascular cell proliferation and migration. Administration of serine elastase inhibitors reduces experimentally induced vascular disease. The ability to mount an intrinsic anti-elastase response may, therefore, protect against intimal/medial thickening after vascular injury. To investigate this, we showed that wire-induced endothelial denudation of the carotid artery is associated with transient elevation in elastase activity and confirmed that this is abolished in transgenic mice overexpressing the serine elastase inhibitor, elafin, targeted to the cardiovascular system. Ten days after injury, nontransgenic littermates show vessel enlargement, intimal thickening, increased medial area and cellularity, and 2-fold increase in tenascin. Injured vessels in transgenic mice become enlarged but are otherwise similar to sham-operated controls. Injury-induced vessel wall thickening, which is observed only in nontransgenic mice, is related to foci of neutrophils and macrophages, in addition to smooth muscle cells that fail to stain for alpha-actin and are likely dedifferentiated. Our study therefore suggests that a major determinant of the vascular response to injury is the early transient induction of serine elastase activity, which leads to cellular proliferation and inflammatory cell migration.

Nitric oxide (NO) reduces the severity of pulmonary vascular disease in rats as do elastase inhibitors. We therefore hypothesized that NO inhibits elastase by suppressing mitogen-activated protein kinases that trans-activate AML1B, a transcription factor for elastase. We used cultured pulmonary artery smooth muscle cells in which serum-treated elastin (STE) induces a > threefold increase in elastase activity as evaluated by solubilization of [(3)H]-elastin. NO donors (SNAP and DETA NONOate) inhibited elastase in a dose-dependent manner as did a cGMP mimetic (8-pCPT-cGMP). SNAP inhibition of elastase was reversed by coadministration of a cGMP-PKG inhibitor (Rp-8-pCPT-cGMP). The STE-induced increase in phospho-ERK was suppressed by NO donors and the cGMP mimetic, and reversed by cGMP-PKG inhibitor, as was expression of AML1B and DNA binding in nuclear extracts. A concomitant increase in p38 phosphorylation was also inhibited by SNAP, but whereas MEK inhibitor (PD98059) suppressed elastase and AML1B-DNA binding, a p38 inhibitor (SB202190) did not. Our study uniquely links NO with inhibition of elastase-dependent matrix remodeling in vascular disease by suggesting a cGMP-PKG-related mechanism suppressing ERK-mediated partitioning of AML1B in nuclear extracts.

Ductus arteriosus intimal cushion formation is characterized by fibronectin-dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthesis in ductus SMC is regulated by the interaction of LC-3, a microtubule-associated protein, with an AU-rich element (ARE) in the 3'-untranslated region of fibronectin mRNA, facilitating its recruitment to polyribosomes for translation. Since nitric oxide (NO) is implicated in posttranscriptional gene regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis. NO production was sevenfold higher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronectin synthesis by approximately 45-50% (P<0.05), whereas the NO donor, SNAP, increased ductus fibronectin synthesis approximately onefold (P<0.05); neither agent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP increased and L-NMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to the fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production regulate the posttranscriptional increase in fibronectin synthesis required for SMC motility.

Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.

Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. In previous studies we showed that the presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Moreover, we demonstrated that denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. In the present study, we examine the pathway by which beta3 integrins stimulate expression of tenascin-C, and define a promoter sequence that is critical for its induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, we transfected A10 cells with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct and showed that, relative to native collagen, its activity is increased on denatured collagen. Next, to identify regions of the promoter involved, we examined a series of tenascin-C promoter constructs with 5' deletions and showed that denatured collagen-dependent promoter activity was retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element was suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. Our data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression.

Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.

In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of approximately 2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells.

We previously reported that serine elastase activity is induced in cultured porcine pulmonary artery (PA) smooth muscle cells (SMC) following serum stimulation by a mechanism involving adhesion of elastin to an elastin binding protein and tyrosine kinase activity. The present study demonstrates that a PA endothelial cell factor also promotes a fourfold increase in elastin adhesion to PA SMC and a twofold increase in serine elastase activity. The mechanism involves tethering of the factor to SMC, since [3H]-elastin pre-incubated with serum or endothelial cell (EC)-conditioned medium or SMC pre-treated with serum accelerates binding of elastin and tyrosine-kinase related elastase activity. The serum factor appears to interact with integrins as elastase induction is partially inhibited by RGD peptides. The elastase-inducing properties of serum could not, however, be attributed to several RGD-containing proteins. While a 120 kD fibronectin fragment partially reproduced the effect, it was not found in the serum fraction containing elastase-inducing activity. Instead, a 27 kD serum protein was enriched by elastin affinity chromatography, identified as apolipoprotein (Apo) A1 by microsequence analysis, and found to have about 50% of the elastase-inducing activity of serum. Elastase induction is inhibited by actinomycin and cycloheximide, suggesting a requirement for mRNA transcription and protein synthesis. Our results suggest a novel cell-extracellular matrix interaction whereby a soluble factor, in this case a lipoprotein, binds and tethers a matrix component to the cell surface and induces tyrosine kinase-dependent transcription of mRNA culminating in substrate proteolysis.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

Tenascin-C, an extracellular matrix glycoprotein prominent during tissue remodeling, has been linked to cell migration, proliferation, and apoptosis. To determine its potential role in the pathobiology of pulmonary hypertension, we compared tenascin expression in adult and infant rat pulmonary arteries (PAs) after injection of the toxin monocrotaline. Immunohistochemistry, in situ hybridization, and Northern blot analysis demonstrated induction of tenascin in adult rat central and peripheral PA. Tenascin was not, however, detected in infant vessels, which show spontaneous regression of vascular lesions. To determine a function for tenascin, we correlated its expression with evidence of apoptosis and cell proliferation using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and 5-bromo-2'-deoxyuridine labeling, respectively. Apoptosis was observed only in the adult rat PA endothelial cell layer, preceding the induction of tenascin, which colocalized both temporally and spatially with proliferating smooth muscle cells (SMCs). A cause-and-effect relationship was documented in cultured rat PA SMCs, where tenascin promoted growth in response to basic fibroblast growth factor and was a prerequisite for epidermal growth factor-induced proliferation. These data provide novel functional information suggesting that endothelial cell apoptosis precedes progressive pulmonary hypertension and that induction of tenascin may be critical to growth factor-dependent SMC proliferation.

We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL)-1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL-1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production.

There is increasing evidence that extracellular matrix (ECM)-degrading proteinases contribute to the process of medial hypertrophy and neointimal proliferation in pulmonary vascular diseases. However, little is known about how proteinases, specifically elastases, induce vascular smooth muscle cell (SMC) hyperplasia. Our objective was to determine whether exogenous human leukocyte elastase (HLE), as well as endogenous vascular elastase, could release basic fibroblast growth factor (bFGF), a potent mitogen stored in the ECM surrounding SMCs. Cultured ovine and porcine pulmonary artery SMC were pre-incubated with [125I]-bFGF. After removal of unbound [125I]-bFGF, administration of HLE (0-1.0 microgram /ml, 1 h) resulted in a concentration-dependent accumulation of [125I]-bFGF in the conditioned medium, mirrored by depletion from the ECM. The serine elastase inhibitor elafin blocked this HLE-mediated action. Assessment by Western immunoblotting further demonstrated that HLE evoked the release of ECM-bound endogenous bFGF. When incubated with serum-starved SMC, conditioned medium from HLE-treated cells stimulated [3H]-thymidine incorporation, a feature neutralized by bFGF antibodies. In addition, SMC exposed to serum treated elastin (STE), previously shown to stimulate endogenous vascular elastase, liberated bioavailable bFGF from ECM stores, as determined by autoradiography, Western immunoblotting, and stimulation of DNA synthesis and SMC proliferation. Chondroitin sulfate, an inhibitor of STE-induced elastase activity, attenuated the release of bFGF. Our studies demonstrate that HLE, secreted by inflammatory cells, and endogenous vascular elastase release matrix-bound bFGF, suggesting a mechanism whereby elastases, through degradation of ECM, induce SMC proliferation associated with progressive vascular disease.

We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-1 beta and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-1 beta was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-1 beta and tumor necrosis factor (TNF)-alpha has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-alpha expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-1 beta, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-alpha antibodies, and exogenous TNF-alpha upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-alpha-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-1 beta antibodies. Likewise, IL-1 beta-stimulated fibronectin synthesis was downregulated to control levels when TNF-alpha neutralizing antibodies were added. Combining TNF-alpha and IL-1 beta enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-1 beta and TNF-alpha activity and provide the first demonstration of a 'cytokine loop' modulating matrix production.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

In previous studies, we related increased elastolytic activity in pulmonary arteries (PA) with endothelial injury to the later development of PA hypertension in rats. As the mechanism causing the increased PA elastase was unknown, we hypothesized that serum factors which are accessible to vascular smooth muscle cells (SMC) following endothelial injury stimulate their elastolytic activity. To test this, we developed an in vitro assay in which we added [3H]-elastin to cultured vascular SMC after 24 h serum starvation and monitored elastolysis following a further 24 h incubation with fetal bovine serum (FBS). We observed that serum induced increased elastolytic activity in both PA and aorta-derived SMC but not in endothelial cells or SMC with low basal levels of elastolytic activity. Maximum stimulation of SMC elastolytic activity occurred with a concentration as low as 1% FBS and despite elastase inhibitors in serum, suggesting that the activity is confined to the immediate pericellular region where enzyme concentration is high. Serum-stimulated elastolytic activity was not reproduced by growth factors or cytokines known to be associated with vascular disease or to induce release of elastases in other cells. The serum inducing elastolytic activity was heat and acid labile. It was associated with increased elastin adhesion to the 67 kD elastin binding protein on SMC surfaces and was prevented by tyrosine kinase inhibitors but not protein kinase C or A inhibitors. Our studies therefore suggest a mechanism whereby serum induction of SMC elastase requires signalling through the elastin binding protein and activation of tyrosine kinase.

Background: We previously identified in piglet cardiac allografts an immunoinflammatory response in coronary arteries in which increased fibronectin regulated by interleukin-1 beta was associated with early evidence of intimal thickening. In the present study, we used rabbits to assess whether acute neointimal formation after cardiac transplantation was reduced by blockade of tumor necrosis factor (TNF)-alpha, which modulates interleukin-1 beta, or by cyclosporine A., Methods and Results: Sixteen rabbits underwent heterotopic cardiac transplantation and were given saline, TNF-soluble receptor (sr), or cyclosporine A. In host hearts from saline- or TNFsr-treated groups, few coronary arteries (approximately 13% to 16%) had intimal thickening, whereas values were higher in the cyclosporine A-treated group (approximately 30%). In donor hearts from the saline-treated group, however, approximately 68% of vessels had intimal thickening versus approximately 32% in TNFsr- and approximately 30% in cyclosporine A-treated groups (P < .01 for both). Severity of intimal thickening assessed quantitatively as percent vessel area was approximately 38% in the saline-treated group but reduced in TNFsr- and cyclosporine A-treated groups to approximately 22% and 18%, respectively (P < .01 for each). Immunohistochemistry revealed increased staining for major histocompatibility complex II, T cells, interleukin-1 beta, TNF-alpha, and fibronectin in donor coronary arteries from saline-treated animals when compared with TNFsr- and cyclosporine A-treated animals. Grade 3 myocardial rejection was observed in both saline- and TNFsr-treated groups, but only grade 1 was apparent in the cyclosporine A-treated group., Conclusions: In vivo blockade of TNF-alpha suppresses the acute development of neointimal formation by selectively reducing the vascular immunoinflammatory reaction and accumulation of fibronectin, whereas cyclosporine A suppresses both the myocardial and the vascular immune reaction.

We established the identity of a 52-kDa protein secreted by fetal lamb ductus arteriosus (DA) smooth muscle cells (SMC) and suggest how it might be related to structural changes unique to DA development, i.e. reduced assembly of elastic laminae and associated formation of intimal cushions. We produced a monoclonal antibody (HI-20) to the 52-kDa protein and observed, by electron microscopy, immunogold labeling of elastin in both DA and aorta vessel walls. Western immunoblotting showed that HI-20, as well as antibodies to tropoelastin, reacted with the 52-kDa protein secreted by DA SMC, as well as with 68-kDa tropoelastin. The highly specific antibody to the carboxyl-terminal sequence of tropoelastin failed, however, to recognize the 52-kDa protein, although it reacted well with the 68-kDa tropoelastin. Amino acid analysis and sequencing data confirmed the identity of the affinity-purified 52-kDa protein as truncated tropoelastin with an intact amino terminus. Cell-free translation of mRNA extracted from DA and aorta SMC produced a 68-kDa, but not a 52-kDa, immunoprecipitated tropoelastin. When DA and aorta SMC were pulsed with [14C]valine, we immunoprecipitated, after only a 15-min chase, both 68-kDa and 52-kDa tropoelastin from cell extracts of DA SMC, but only the 68-kDa tropoelastin was present in aorta SMC. There was no evidence of proteolytic degradation of radiolabeled aorta 68-kDa tropoelastin to a 52-kDa species when mixed with DA SMC conditioned medium. This suggests that the 52-kDa tropoelastin is the result of cell-associated processing or degradation of an original 68-kDa product of translation. Furthermore, pulse-chase experiments showed initial secretion of equivalent amounts of 68-kDa and 52-kDa tropoelastins by cultured DA SMC with increasing accumulation of the 52-kDa species, suggesting its impaired insolubilization. The production, in high concentration, of a 52-kDa tropoelastin product that lacks the carboxyl terminus, may prevent its alignment on the microfibrillar scaffold, resulting in abnormal assembly of elastic laminae in the DA. The accumulation of this soluble tropoelastin may be associated with the previously described property of chemotaxis resulting in the increased SMC migration into the subendothelium associated with DA intimal thickening.

Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in N-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a three-dimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.

Background: In previous studies we established that there are developmentally regulated increases in endothelial hyaluronan (HA) and heparan sulfate (HS), and smooth muscle cell fibronectin (FN) related to the formation of intimal cushions, structures essential to the postnatal closure of the ductus arteriosus (DA). In this report, we investigated the mechanisms underlying these features to ascertain whether they were independently or coordinately regulated., Experimental Design: We determined by assessing HA polymer size and by pulse labeling with [3H]glucosamine whether the increased glycosaminoglycans (GAGs) incorporated in DA compared with aorta (Ao) endothelial cell matrices reflected increased synthesis of HA and HS rather than decreased degradation. We assessed whether transforming growth factor-beta (TGF-beta) may be responsible for the increased DA endothelial GAGs and smooth muscle FN production by confirming the presence of TGF-beta in DA tissue using immunohistochemistry and by assessing the effect of adding neutralizing antibodies to the cell cultures. We next determined whether the level of regulation of the increase in FN in DA smooth muscle cells was transcriptional or post-transcriptional by relating protein synthesis to steady state mRNA levels and to mRNA levels after serum stimulation. Using northern blot analyses with specific probes, we also explored the possibility that the FN produced by the DA and Ao was qualitatively different in the proportion of isoforms containing the V95+ region associated with secretion or the EIIIB+ region that has been related to migration., Results: We observed that HA polymer size produced by DA and Ao endothelial cells was similar and we further verified using pulse labeling that the increase in DA compared with Ao endothelial GAGs reflected increased synthesis of HA and HS rather than decreased degradation. There was increased immunostaining for TGF-beta in DA compared with Ao tissue and we showed that TGF-beta neutralizing antibodies reduced synthesis of GAGs by the DA endothelial cells to the level of that seen in the Ao cells, but did not reduce DA smooth muscle cell FN synthesis. The increase in FN synthesis by DA compared to Ao smooth muscle cells was not associated with increased levels of steady-state mRNA for FN. Furthermore, following serum-stimulated increases in FN mRNA, the DA yielded greater amounts of FN protein compared to Ao smooth muscle cells. The increased FN production by the DA smooth muscle cells could not be attributed to a relative lack of degradation of FN protein or mRNA, or to qualitative differences which might influence secretion, as both cell types contained similar proportions of EIIIB+ and V95+ isoforms of FN., Conclusions: These results would suggest that, in contrast to the TGF-beta dependent increase in DA endothelial GAG synthesis, the increase in DA smooth muscle FN synthesis arises through differences in post-transcriptional regulation that are likely independent of TGF-beta.

In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.

In the late gestation fetal lamb ductus arteriosus (DA), intimal proliferation is observed, characterized by smooth muscle migration and proliferation in the subendothelium. The nature of changes in the endothelial and smooth muscle extracellular matrix associated with the development of this feature are not known. We assessed the production of glycoproteins (fibronectin, laminin, and type IV collagen) and glycosaminoglycans (GAGs) (hyaluronic acid, heparan sulfate, and chondroitin sulfate) in endothelial and smooth muscle cells harvested from the DA, aorta (Ao), and pulmonary artery of fetal lambs at 100 days gestation, before the appearance of DA intimal proliferation, and at 138 days, when well-developed intimal 'cushions' are seen. In passage 3 cells, glycoprotein synthesis was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after 48 hours incubation with [35S]methionine, and GAGs were assessed by labeling with [3H] glucosamine and separation on DEAE ion-exchange high performance liquid chromatography. Analyses were carried out on culture medium, cell layer, and solubilized matrix. Fibronectin secretion by DA smooth muscle cells from 100-day lambs was found to be twice that of Ao or pulmonary artery cells. No significant differences were seen in smooth muscle cells from 138-day lambs or when comparing endothelial cells from each of the vascular sites at both gestational ages. As well, there were no DA-specific differences in laminin or type IV collagen. No significant differences in endothelial GAG secretion were observed comparing each vascular site at both gestational ages. Analysis of endothelial-derived matrices, however, revealed increased incorporation of hyaluronic acid in the DA from 100-day lambs, 10-fold that of the pulmonary artery and Ao, and increased heparan sulfate. These differences were still present in cell matrices from late gestation animals, but were less marked. No differences in GAGs were seen when comparing smooth muscle cells. Incubation of 100-day DA and Ao smooth muscle cells with endothelial conditioned medium however, resulted in a 2-fold increase in chondroitin sulfate in DA, compared with Ao. These results indicate that distinct, developmentally regulated patterns of extracellular matrix production are related to vascular site and specific features appear to precede intimal proliferation in the DA.

Pulmonary artery and aorta endothelial and smooth muscle cells harvested from near term Rambouillet lambs were cultured. To determine whether cultured endothelial cells produce a factor which alters the shape and alignment of smooth muscle cells, rhodamine phalloidin staining of F-actin filaments was carried out, as well as time-lapse cinematography. We were able to demonstrate, with pulmonary artery but not with aortic cells in culture, an endothelial-derived factor which causes the smooth muscle cells to migrate and align in uniform direction. The effect could not be enhanced by making the endothelial cells hypoxic and was similar to that produced by exposing smooth muscle cells to KCl or directly to hypoxia.

During late gestation, intimal cushions form in the ductus arteriosus (DA) and these cause the vessel to close when it constricts in the postnatal period. The formation of intimal cushions suggests highly specialized functions of DA endothelial and smooth muscle cells. To investigate these properties, we established, from fetal lambs on Day 138 of a 148-day term gestation, primary cell cultures of DA endothelium and smooth muscle and compared them to cells derived from the adjacent pulmonary artery and aorta. Purity of the endothelial cell cultures from each vascular site was assessed by the contact inhibited "cobblestone" monolayer phenotype, by positive immunofluorescence for factor VIII and by angiotensin converting enzyme activity. Purity of smooth muscle cell cultures at each vascular site was assessed by the "hills and valleys" phenotype and by positive immunofluorescence with a smooth muscle actin specific monoclonal antibody. Endothelial and smooth muscle cells had different growth curves, ultrastructural features, and protein profiles on single and two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE), but vascular sites were similar. To further determine whether differences related to DA origin were indeed present, endothelial and smooth muscle cells from all three vascular sites were incubated with the radiolabeled amino acids [14C]leucine, [14C]proline, and [14C]valine and the proteins in both the cells and the conditioned medium were analyzed by autoradiography after SDS-PAGE. A dense band corresponding to a 42-kDa protein was observed in valine-labeled DA endothelial cells and conditioned medium and a 52-kDa protein was observed in the conditioned medium of leucine-labeled DA smooth muscle cells only. Further isolation and characterization of these endothelial and smooth muscle proteins will be necessary to determine whether they are related to the mechanism of intimal cushion formation in the late gestation DA or are present abnormally in association with the intimal proliferation observed in pulmonary and systemic vascular disease.