generates a decrease in diffusing capacity for carbon monoxide (DLCO) and forced vital capacity (FVC) that corresponds to the forced expiration (Clements, 2004).
The thickening of the pulmonary arteries is a result of several vascular changes that affect all three layers (i.e., intima, media and adventitia) of the pulmonary vessel. Each cell type (endothelial, smoothmuscle and fibroblast) in the pulmonary vascular wall plays a specific role in the pathogenesis (Humbert, 2008). Endothelial cell (EC) injury is the critical early event in SSc-associated pulmonary vascular disease. It leads to alterations in endothelial function with increased production of vasoconstrictor mediators, such as endothelin-1 (ET-1), and decreased synthesis of prostacyclin and nitric oxide (NO) (Varga, 2002). In normal individuals, vasoactive molecules produced from EC adapt the pulmonary vascular smoothmusclecells (VSMCs) tone to the actual needs of the organism and keep the smoothmuscle in a state of relaxation. In PAH, the imbalanced productions of vasoactive mediators lead to increased pulmonary vascular reactivity and abnormal vasoconstriction (Muller-Ladner, 2009).
Abstract Leukotrienes are pro-inflammatory mediators that are locally produced in coronary atherosclerotic plaques. The response induced by cysteinyl leukotrienes (CysLT) in human coronary arteries may be altered under pathological condi- tions, such as atherosclerosis. The aim of the present study was to elucidate cysteinyl leukotriene signaling in vascular smoothmusclecells (SMCs) and the effects of inflammation on this process. Immunohistochemical analysis of human carotid endarterectomy samples revealed that the CysLT 1
levels in patients with Group III PH ( Garcia-Morales et al., 2016 ). Experiments using the ADORA2B antagonist (GS-6201) or full Adora2b knock-out mice revealed that genetic deletion or pharmacological inhibition of Adora2b subdued both the fibrotic deposition and the development of PH in a mouse model of chronic bleomycin (BLM)-induced lung fibrosis and PH ( Karmouty-Quintana et al., 2012 ). Further studies revealed that conditional deletion of Adora2b from the myeloid lineage resulted in a reduction in fibrotic deposition and the absence of hallmarks of PH, including thickening of the vascular wall and elevated right ventricle systolic pressure (RVSP), in mice with conditional deletion of Adora2b in myeloid cells ( Karmouty- Quintana et al., 2015 ). In experiments using pulmonary artery smoothmusclecells (PASMC), studies have shown that activation of ADORA2B can lead to increased expression of hyaluronan synthase (HAS) isozymes 1 and 2, enhancing levels of hyaluronan ( Karmouty-Quintana et al., 2013a ), the major glycosaminoglycan in the lungs that when fragmented has been implicated in modulating PH and lung fibrosis ( Karmouty-Quintana et al., 2013a; Collum et al., 2017b ). However, expression levels of CD39, CD73, ADA, and adenosine receptors in PAH has not yet been evaluated. In addition, the effects of conditional deletion of ADORA2B from vascular smoothmusclecells on the development of PH have not yet been determined. Here, we have assessed expression levels of mediators involved in the generation and metabolism of adenosine in PAH and evaluated the effects of conditional deletion of ADORA2B in vascular smoothmusclecells using the transgelin (Tagln) promoter, also known as smoothmuscle protein 22-alpha promoter. In these in vivo experiments, mice were exposed to two distinct models of PH: the chronic hypoxia-SUGEN (HX-SU) model of PH and the BLM model of lung fibrosis and PH. In addition, we performed cell culture studies using human isolated PASMCs to identify ADORA2B-mediated mechanisms leading to PH.
Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease in children that leads to early death. Smoothmusclecells (SMCs) are the most affected cells in HGPS individuals, although the reason for such vulnerability remains poorly understood. In this work, we develop a micro ﬂuidic chip formed by HGPS-SMCs generated from induced plur- ipotent stem cells (iPSCs), to study their vulnerability to ﬂow shear stress. HGPS-iPSC SMCs cultured under arterial ﬂow conditions detach from the chip after a few days of culture; this process is mediated by the upregulation of metalloprotease 13 (MMP13). Importantly, double-mutant Lmna G609G/G609G Mmp13 −/− mice or Lmna G609G/G609G Mmp13 +/+ mice trea- ted with a MMP inhibitor show lower SMC loss in the aortic arch than controls. MMP13 upregulation appears to be mediated, at least in part, by the upregulation of glycocalyx. Our HGPS-SMCs chip represents a platform for developing treatments for HGPS individuals that may complement previous pre-clinical and clinical treatments.
we found concentration-dependent inhibition by CFTR inh -172 of the bronchodilation induced by the CFTR
activators MPB after muscarinic stimulation. This phar- macological evidence is in favour of an unexpected role of CFTR in bronchodilation. It is well known that airway smoothmuscle relaxation is brought about predomi- nantly by stimulation of adenyl cyclase-coupled receptors (e.g. β2-adrenoceptor) resulting in elevation of cell cyclic adenosine monophosphate content. Importantly, this sig- nalling pathway is central in activating CFTR-mediated chloride transport in epithelial [7,21], aortic , and air- way smoothmusclecells. Taken together these results illu- minate a direct implication for CFTR in the bronchodilation of the rat trachea.
In the second part of the work, we analyzed the functional contribution of these PDE isoforms in controlling intracellular cAMP concentration. For this purpose, we took advantage of the FRET technique using an Epac1-based sensor which allows a real-time monitoring of cAMP concentrations in living cells [13,14]. This approach has been applied extensively in several cell types, including cell lines  or freshly isolated cells such as cardiomyocytes , but rarely in vascular smoothmusclecells [35,36]. In mice aorta SMCs maintained one week in culture after dissociation, Iso increased intracellular cAMP with an EC 50 of about 40 nM . In our study, we also
Vascular remodeling due to the hyperproliferation and hypertrophy of vascular smoothmusclecells (VSMCs) is central in the development of hypertension. VSMCs from spontaneously hypertensive rats (SHR) exhibit hyperproliferation and overexpression of cell cycle proteins. Resveratrol, a natural polyphenolic compound found in the skin of grapes, is implicated in several vasoprotective effects. Resveratrol has also been reported to attenuate angiotensin II-induced VSMCs proliferation. However, it was not elucidated if resveratrol could also inhibit the hyperproliferation of VSMCs from SHR. The present study was undertaken to investigate if resveratrol could attenuate the hyperproliferation of VSMCs and explore the underlying molecular mechanisms responsible for this effect. Methods: For this study, aortic VSMCs from 14-week-old SHR and Wistar-Kyoto (WKY) rats were used. The proliferation of VSMCs was determined by [ 3 H] thymidine incorporation and the levels of proteins were determined by Western blotting techniques. Results: VSMCs from SHR exhibit a hyperproliferation which was attenuated by resveratrol. The overexpression of cyclin D1, cyclin E, cyclin dependent kinase 4 (CDK4), cyclin dependent kinase 2 (CDK2), phosphorylated retinoblastoma protein (pRb), Giα-3, Giα-2 proteins and enhanced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT in VSMCs from SHR were attenuated by resveratrol. Furthermore, resveratrol also inhibited the increase of the superoxide anion, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, overexpression of NADPH oxidase 2 (NOX2)/NADPH oxidase 4 (NOX4) and P47 phox proteins, and the increased phosphorylation of epidermal growth factor receptor (EGF-R), insulin-like growth factor 1 receptor (IGF-1R), and c-Src to control levels. These results suggest that resveratrol through the inhibition of reactive oxygen species (ROS) and ROS-mediated transactivation of growth factor receptors, c-Src, mitogen-activated protein kinases (MAPK)/Phosphoinositide-3 kinase (PI3K), attenuates the overexpression of Giα and cell cycle proteins and results in the attenuation of hyperproliferation of VSMCs from SHR.
regulation (death or survival), cell motililty (migration, adhesion), and extracellular matrix turnover (secretion and degradation) (47). Endothelial dysfunction has been suggested to be the initial step that prompts susbsequent events in the progression of vascular dysfunction (48). In fact, the adluminal position of endothelial cells allows a direct contact with the bloodstream. This supports the idea that, hypertension and the other determinants of CVD such as hyperglycemia and dyslipidemia, by directly affecting endothelial cell physiology, are able to cause vascular damage (49). Deleterious consequences of chronic metabolic disturbances also involve aberrant expansion of inflammatory cells via hyperplasic and migratory responses. These inflammatory events participate in vascular lesion formation, an ultimate event in the atherogenic process (37). Because of the importance of the role played by inflammatory cells and determinants of innate immunity in vascular disease, it is also being considered as an immune disorder. This is supported by studies where animals bearing a deficiency in T or B lymphocytes (50), monocytes or macrophage (51), failed to develop vascular injury in disease promoting conditions. Finally, it is well documented that the pathophysiological responses exhibited by vascular smoothmusclecells (VSMC) underlie the structural changes observed in the vessel wall and further connect inflammatory responses and endothelial dysfunction to the development of vascular damage (52). Thus, together, individual properties beared by cells within the vasculature contribute to the pathogenesis of vascular damage. Accordingly, many vascular cell types in culture have been considered as in vitro models for the study of vascular disease. In the context of this thesis however, because of the central contribution of VSMC to vessel stiffness and reactivity, further description of the molecular basis of vessel remodeling will be focused on the signal transduction that control their physiology.
The discovery of endothelin (ET), the potent vasoconstrictor, surfaced only two decades ago and the endothelin peptide family has now evolved into an intricate system. Endothelin-1 (ET-1) is the most predominant isoform synthesized by the vasculature, mainly by the vascular endothelium and smoothmusclecells, and is accountable for the majority of the pathobiological effects exerted by the endothelin peptide family. In the vascular system, ET-1 mainly exerts a basal vascular tone regulation. A long term upregulated ET-1 system has been suggested to contribute to the deterioration of vascular function that leads to the development of vascular diseases. ET-1 exerts its action through the activation of its endothelin receptors, ETA and ETB, but ETA is mainly responsible in the contribution of ET-1 to the pathogenesis of vascular abnormalities, such as hypertension, atherosclerosis, restenosis, and other cardiovascular diseases. Based on recent research, ET-1 receptor antagonists exhibit inhibition of ET-1-induced functional and structural alterations in the vasculature. Nevertheless, ET-1 mediates its pleiotropic actions through the activation of several signaling pathways and further investigation is required to provide a better knowledge of these ET-1 signal transducing pathways for designing specific therapeutic agents directed against critical components of the signaling systems implicated in pathological contribution of ET-1-induced vascular remodeling.
On the basis of both experimental and clinical data, we found that estrogens have been proposed to exert several
protective arterial effects. In particular, 17β-estradiol (E2), the main endogenous estrogen, has a dual beneficial effect on the 2 facets of vascular healing after angioplasty because it both accelerates endothelial regrowth and inhibits the pro- liferation of vascular smoothmusclecells (VSMC), which otherwise leads to the narrowing of the arterial lumen (reste- nosis). 4 Consistent with these functions, E2 has been shown
Summary. Human angiogenin is a plasma protein with angiogenic and ribonucleolytic
activities. Angiogenin inhibited both DNA replication and proliferation of aortic smoothmusclecells. Binding of 125I-angiogenin to bovine aortic smoothmusclecells at 4°C was specific, saturable, reversible and involved two families of interactions. High-affinity binding sites with an apparent dissociation constant of 2 x 10-10 M bound 1 x 104 molecules per cell grown at the density of 3 x 104/cm2. Low-affinity binding sites with an apparent dissociation constant of 1 x 10-7 M bound 4 x 106 molecules/cell. High-affinity binding sites decreased as cell density increased and were not detected at confluence. 125I- angiogenin bound specifically to cells routinely grown in serum-free conditions, indicating that the angiogenin-binding components were cell-derived. Affinity labelling of sparse bovine smoothmusclecells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250-260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Altogether these data suggest that cultured bovine and human aortic smoothmusclecells express specific receptors for human angiogenin.
Physique des Polyme`res, Institut Carnot, CIRIMAT UMR 5085, Universite´ Paul Sabatier, Tolouse, France; and ‡ Cardiovascular Research Center, CSIC-ICCC, IIB-Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
ABSTRACT Aggregated low-density lipoprotein (agLDL), one of the main LDL modifications in the arterial intima, contributes to massive intracellular cholesteryl ester (CE) accumulation in human vascular smoothmusclecells (VSMC), which are major producers of elastin in the vascular wall. Our aim was to analyze the levels, physical structure, and molecular mobility of tropoe- lastin produced by agLDL-loaded human VSMC (agLDL-VSMC) versus that produced by control VSMC. Western blot analysis demonstrated that agLDL reduced VSMC-tropoelastin protein levels by increasing its degradation rate. Moreover, our results demonstrated increased levels of precursor and mature forms of cathepsin S in agLDL-VSMC. Fourier transform infrared anal- ysis revealed modifications in the secondary structures of tropoelastin produced by lipid-loaded VSMCs. Thermal and dielectric analyses showed that agLDL-VSMC tropoelastin has decreased glass transition temperatures and distinct chain dynamics that, in addition to a loss of thermal stability, lead to strong changes in its mechanical properties. In conclusion, agLDL lipid loading of human vascular cells leads to an increase in cathepsin S production concomitantly with a decrease in cellular tropoelastin protein levels and dramatic changes in secreted tropoelastin physical structure. Therefore, VSMC-lipid loading likely determines alterations in the mechanical properties of the vascular wall and plays a crucial role in elastin loss during atherosclerosis.
calcification-induced aortic rings of rats and clinical biomarkers [ 15 ]. To date, three studies di- rectly addressed the effect of magnesium on VC of VSMC in vitro. Here, magnesium signifi- cantly reduced induced calcification both in bovine as well as human primary VSMC,
downregulated known pro-calcification and upregulated anti-calcification markers towards re- storing more physiological expression levels seen in uncalcified controls [ 16 , 17 , 18 ]. Moreover, an active, cellular, inhibitory role was attributed to magnesium as it was only able to exert its protective effect on living cells [ 17 ]. These findings do not exclude a potential passive role for Mg 2+ ions in the onset of calcification, because in the presence of Mg 2+ cumulating evidence shows that magnesium can have an inhibitory effect on hydroxyapatite formation and precipi- tation. Earlier in vitro work shows that magnesium is able to stabilize amorphous calcium phosphate and inhibits the formation of hydroxyapatite as well as calcium pyrophosphate de- hydrate and calcium-acidic phospholipid-phosphate complexes [ 19 – 23 ]. In the literature, bio- logical calcium phosphate crystals are often referred as hydroxyapatite leading this term to designate many complex compounds. Indeed, calcium phosphate hydroxyapatite is considered as a model compound for biological mineralization although its ideal formula has not actually been found in vivo. The composition of calcium phosphate apatite crystals has been shown to vary over a wide range due to the possibilities of anionic and cationic substitutions and the ex- istence of different type of ion vacancies [ 24 ].
qRT-PCR. Following MACS, cells were immediately lysed and total RNA was extracted using the RNeasy ® Micro Kit (Qiagen). Reverse transcription was performed on 100 ng of total RNA using random primers (cat # N8080127) and the Superscript II Reverse Transcriptase (cat # 18064022) following the supplier’s recommenda- tions (Life Technologies). For qPCR, 8 µL of cDNA diluted 1/20 was mixed with 10 µL of SYBR Green fluores- cence detection solution (Thermo Fisher Scientific) and 1 µL of each primer (Eurogentec) (see Supplementary Table S1 for primers’ sequence). qRT-PCR was performed in an ABI Prism ® 7900HT Sequence Detection System (Applied Biosystems) using the following program: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of amplification (15 sec at 95 °C, 45 sec at 60 °C). All primers had amplification efficiency close to 100% as checked by the analysis of standard dilution curves (slope close to −3.33) so that the relative expression of a gene X to the housekeeping gene Tbp could be calculated by 2 −ΔCt where ΔCt = Ct(X) − Ct(Tbp) and Ct is the threshold cycle value.
2.6.6 Immunoblot analyses.
350,000 VSMCs were seeded in 6-well plates in DMEM/10%FBS. Quiescent cells were stimulated with 200 µg/mL purified IgG  for 5 minutes, at 37°C under 5% CO 2 . After
5 minutes, cells were gently washed two times with ice-cold PBS and whole cell extracts were prepared using Triton-X lysis buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM β-glycerophosphate, 1 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL leupeptin, 1 µM pepstatin A, 2 µg/mL aprotinin, 1% Triton-X-100, 10% glycerol) for 30 min at 4˚C. Lysed material was then centrifuged at 13,000 x g for 10 min and the supernatant collected. Equal amounts of lysate proteins (20-50 µg) were loaded on 7.5 or 10% polyacrylamide gels and subjected to SDS- PAGE. In some Western blot experiments, cellular extracts were divided and used in parallel. Proteins were transferred to nitrocellulose membranes in 25 mM Tris, 192 mM glycine and 20% methanol using a Bio-Rad Transblot Cell transfer apparatus. Immunoblotting with each antibody was carried out according to manufacturer instructions.