Localization of elastin mRNA and TGF-β1 in rat aorta and caudal artery as a function of age

Several in vitro studies have previously demonstrated that the addition of TGF-β to aortic smooth muscle cells or skin fibroblasts stimulates elastin synthesis. It is not clear however whether, in vivo, TGF-β participates in the regulation of elastin synthesis, especially in physiological conditions. The aim of our study was to explore the localization of elastin mRNA and TGF-β1 in the rat thoracic aorta (an elastic artery) and caudal artery (a muscular artery). Elastin mRNA was localized by in situ hybridization and quantified using Northern blot analysis. TGF-β1 was detected using immunohistochemistry. The study was carried out as a function of age (rats of 3, 10, 20, and 30 months). We observed that TGF-β1 immunoreactivity is present predominantly, but not exclusively, at the sites of elastin synthesis as determined by elastin mRNA detection: in smooth muscle cells in the aorta and in endothelial cells in the caudal artery. The ability of exogenously added TGF-β1 (0.001–10 ng/ml) to modulate the steady-state levels of elastin mRNA in primary cultures of endothelial cells, smooth muscle cells, and fibroblasts isolated from the thoracic aorta was also studied. At the highest concentration used, elastin mRNA levels increased 5-fold in endothelial cells and 11-fold in smooth muscle cells. The demonstration that TGF-β1 immunoreactivity is present at the sites of elastin synthesis in the thoracic aorta and in the caudal artery and the observation that TGF-β1 induces an increase in elastin mRNA levels in cultured endothelial cells and smooth muscle cells suggest that TGF-β1 may be implicated, at least in part, in the physiological regulation of elastin gene expression.     

Insulin down-regulates TRAIL expression in vascular smooth muscle cells both in vivo and in vitro

To dissect the effect of hyperinsulinemia versus hyperglycemia on TNF-related apoptosis inducing ligand (TRAIL) expression in the macrovascular district, we measured TRAIL mRNA and protein in four groups of animals: streptozotocin (SZT)-induced diabetic rats, vehicle-treated control animals, diabetic rats treated with insulin and non-diabetic rats treated with insulin. While the aortas of diabetic rats did not show significant differences in TRAIL expression with respect to vehicle-treated control animals, the aortas of both diabetic and non-diabetic rats treated in vivo for 16 days with insulin showed a significant decrease in TRAIL expression with respect to either diabetic and control rats. Moreover, in vitro treatment of both rat and human vascular smooth muscle cells (VSMC) with insulin induced the down-regulation of TRAIL protein. While the addition of recombinant TRAIL to rat VSMC promoted the dose-dependent release of bioactive nitric oxide (NO), this effect was significantly counteracted by pre-exposure of VSMC to insulin. These findings suggest that TRAIL might act as an endogenous regulator of the vascular tone and that chronic elevation of insulin might contribute to the vascular abnormalities characterizing type-2 diabetes mellitus by down-regulating TRAIL expression and activity.     

内质网应激在大鼠低氧高二氧化碳性肺动脉高压中的作用

目的：观察低氧高二氧化碳性肺动脉高压大鼠的肺血管重塑并探讨内质网应激（ERS）在肺动脉高压中的作用。方法：将40只SD大鼠随机分为四组：常氧对照组（N）、低氧高二氧化碳组（HH）、ERS通路抑制剂4-苯基丁酸（4-phenylbutyric acid）组（4-PBA）、ERS通路激动剂衣霉素（tunicamycin）组（TM）,n=10。测量各组大鼠的肺动脉平均压（mPAP）、颈动脉平均压以及右心室肥大指数,免疫荧光α-SMA标记法鉴定各组肺中小动脉平滑肌细胞,电镜观察肺组织及肺中小动脉形态学变化,原位末端标记法（TUNEL）检测各组肺动脉平滑肌细胞的凋亡指数,采用RT-PCR和Western blot分别检测各组大鼠葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、c-Jun氨基末端激酶（JNK）、天冬氨酸特异性半胱氨酸蛋白酶-12（caspase-12）mRNA及蛋白质表达。结果：①与N组相比,HH组、4-PBA组、TM组mPAP、右心室游离壁重量/左心室加心室间隔重量[RV/（LV+S）]、肺动脉管壁面积/管总面积（WA/TA）比值增加（P<0.0 1）,肺动脉管腔面积/管总面积（LA/TA）比值减小（P<0.01）,细胞凋亡指数降低（P <0.05或P<0.01）。ERS相关蛋白质及mRNA的表达量升高,各差异均有统计学意义。②与HH组相比,4-PB A组mPAP和[RV/（LV+S）]、WA/TA值减小（P<0.01）,LA/TA值和细胞凋亡指数上升（P<0.05或P<0.01）,ERS相关蛋白质和mRNA的表达量均下调（P<0.05或P<0.01）;③与HH组相比,TM组mPAP、[RV/（LV+S）]、WA/TA值升高（P<0.05或P<0.01）;肺动脉中膜层增厚,LA/TA值和细胞凋亡指数降低（P<0.01）。ERS相关蛋白质及mRNA的表达量均升高,除GRP78蛋白质表达量无明显变化外,其余各差异均有统计学意义。结论：低氧高二氧化碳诱导的肺动脉高压大鼠肺血管重塑可能与肺动脉平滑肌细胞增殖过度及凋亡过少有关;ERS相关因子（JNK、caspase-12和CHOP）参与低氧高二氧化碳性肺动脉高压的调控。     

Bone morphogenetic protein (BMP) type II receptor is required for BMP-mediated growth arrest and differentiation in pulmonary artery smooth muscle cells

Bone morphogenetic protein (BMP) signals regulate the growth and differentiation of diverse lineages. The association of mutations in the BMP type II receptor (BMPRII) with idiopathic pulmonary arterial hypertension suggests an important role of this receptor in vascular remodeling. Pulmonary artery smooth muscle cells lacking BMPRII can transduce BMP signals using ActRIIa (Activin type II receptor). We investigated whether or not BMP signaling via the two receptors leads to differential effects on vascular smooth muscle cells. BMP4, but not BMP7, inhibited platelet-derived growth factor-activated proliferation in wild-type pulmonary artery smooth muscle cells, whereas neither ligand inhibited the growth of BMPRII-deficient cells. Adenoviral gene transfer of BMPRII enabled BMP4, as well as BMP7, to inhibit proliferation in BMPRII-deficient cells. BMP-mediated growth inhibition was also reconstituted by the BMPRII short isoform, lacking the C-terminal domain present in the long form. BMP4, but not BMP7, induced the expression of osteoblast markers in wild-type cells, whereas neither ligand induced these markers in BMPRII-deficient cells. Overexpression of short or long forms of BMPRII in BMPRII-deficient cells enabled BMP4 and BMP7 to induce osteogenic differentiation. Although signaling via BMPRII or ActRIIa transiently activated SMAD1/5/8, only BMPRII signaling led to persistent SMAD1/5/8 activation and sustained increases in Id1 mRNA and protein expression. Pharmacologic blockade of BMP type I receptor function within 24 h after BMP stimulation abrogated differentiation. These data suggest that sustained BMP pathway activation, such as that mediated by BMPRII, is necessary for growth and differentiation control in vascular smooth muscle.     

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells

BackgroundThe intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs.MethodsFor these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs.ResultsTreatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls.SummaryThese in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.     

Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways.     

Differential effects of TGF-beta1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins. The transforming growth factor (TGF)-beta superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-beta1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38(MAPK) or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-beta1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-beta1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-beta superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.     

Chronic administration of adrenomedullin attenuates hypoxic pulmonary vascular structural remodeling and inhibits proadrenomedullin N-terminal 20-peptide production in rats

Adrenomedullin (ADM) is a novel cardiovascular-active peptide involved in vasodilation, reducing blood pressure and inhibiting vascular smooth muscle cell migration and proliferation. Previous research showed that ADM might be involved in the development of pulmonary hypertension. In this study, we investigated the effect of ADM subcutaneously administered by mini-osmotic pump (300 ng/h) on pulmonary hemodynamics and pulmonary vascular structure in hypoxic rats, as well as the influence of ADM on the proadrenomedullin N-terminal 20-peptide (PAMP) protein and mRNA expressions and its plasma concentrations. The results showed that ADM obviously decreased mean pulmonary artery pressure and the ratio of right ventricular mass to left ventricular plus septal mass in hypoxic rats. Chronic infusion of ADM lessened the muscularization of small pulmonary vessels, attenuated relative medial thickness and relative medial area of pulmonary arteries, and alleviated the ultrastructural changes in pulmonary arteries of hypoxic rats. ADM inhibited the proliferation of pulmonary artery smooth muscle cells, represented by a decrease in the expression of proliferative cell nuclear antigen (PCNA) in the pulmonary artery. Meanwhile, plasma PAMP concentration and the expression of PAMP protein and mRNA by pulmonary arteries in rats of hypoxia with ADM group were markedly decreased compared with those in hypoxic group. The results suggest that ADM ameliorated the development of hypoxic pulmonary vascular structural remodeling. Intramolecular regulation of ADM may play an important role in the regulation of hypoxic pulmonary hypertension by ADM.     

Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.     

Expression of serotonin receptor mRNAs in blood vessels

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1 _β, 5-HT_2A, 5-HT_2B, 5-HT₄, and 5-Ht₇ receptor mRNA to different extents. New splice variants of the porcine 5-HT₄ receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT_{1 β}, 5-HT_2b, and 5-HT₄ receptor mRNA, whereas in smooth muscle cells 5-HT_{1 β}, 5-HT_2A, 5-HT₇, and in some experiments 5-HT_2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.     

Platelet-derived growth factor induces c-fms and scavenger receptor genes in vascular smooth muscle cells.

Vascular smooth muscle cells proliferate and transform to foam cells in the process of atherosclerosis. In the present study, we demonstrated that platelet-derived growth factor (PDGF)-BB induced expression of proto-oncogene c-fms in vascular smooth muscle cells, which normally do not express c-fms, isolated from either human umbilical artery or rabbit aorta. No effect of the protein kinase C activator, phorbol ester, was demonstrated on mRNA expression of c-fms. In contrast, the scavenger receptor activity was induced by both PDGF-BB and phorbol ester. These results indicate that two characteristic genes of monocyte-macrophages were induced by PDGF-BB via the different pathways, and suggest that PDGF-BB plays an important role in initiating phenotypic conversion of smooth muscle cells to macrophage-like cells.     

TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to have protective effects against atherosclerosis. However, whether TRAIL has any effects on expression of macrophage scavenger receptors and lipid uptake has not yet been studied. Macrophage lines RAW264.7 and THP-1, and mouse primary peritoneal macrophages, were cultured in vitro and treated with recombinant human TRAIL. Real-time PCR and western blot were performed to measure mRNA and protein expressions. Foam cell formation was assessed by internalization of acetylated and oxidized low-density lipoproteins (LDL). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. We found that TRAIL treatment increased expression of scavenger receptor (SR)-AI and SR-BI in a time- and dose-dependent manner, and this effect was accompanied by increased foam cell formation. These effects of TRAIL were abolished by a TRAIL neutralizing antibody or in DR5 receptor-deficient macrophages. The increased LDL uptake by TRAIL was blocked by SR-AI gene silencing or the SR-AI inhibitor poly(I:C), while SR-BI blockade with BLT-1 had no effect. TRAIL-induced SR-AI expression was blocked by the inhibitor of p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2 or JNK. TRAIL also induced apoptosis in macrophages. In contrast to macrophages, TRAIL showed little effects on SR expression or apoptosis in vascular smooth muscle cells. In conclusion, our results demonstrate that TRAIL promotes macrophage lipid uptake via SR-AI upregulation through activation of the p38 pathway.     

TRAIL stimulates proliferation of vascular smooth muscle cells via activation of NF-kappaB and induction of insulin-like growth factor-1 receptor

TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) is a multifunctional protein regulating homeostasis of the immune system, infection, autoimmune diseases, and apoptosis. However, its function in normal, nontransformed tissues is not clear. Here we show that TRAIL increases vascular smooth muscle cell (VSMC) proliferation in vitro, effects that can be blocked with neutralizing antibodies to TRAIL receptors DR4 and DcR1. In aortocoronary saphenous vein bypass grafts in vivo, TRAIL co-localizes with VSMC, proliferating cell nuclear antigen, and insulin-like growth factor type 1 receptor (IGF1R) expression but not active caspase-3. TRAIL is required for serum-inducible IGF1R expression, and antisense IGF1R inhibits TRAIL-induced VSMC proliferation. At 1 ng/ml, TRAIL stimulates IGF1R mRNA expression greater than insulin-like growth factor-1 and also activates the IGF1R promoter 7-fold. TRAIL-inducible IGF1R expression requires NF-kappaB activation. Consistent with this, ammonium pyrrolidine dithiocarbamate, a pharmacological inhibitor of NF-kappaB, blocks TRAIL-induced IGF1R expression, and p65 overexpression increases IGF1R protein levels. In addition, NF-kappaB binds a novel TRAIL-responsive element on the IGF1R promoter. Our findings suggest that the biological functions of TRAIL in VSMC extend beyond its role in promoting apoptosis. Thus, TRAIL may play an important role in atherosclerosis by regulating IGF1R expression in VSMC in an NF-kappaB-dependent manner.     

Regulation of TRAIL expression by the phosphatidylinositol 3-kinase/Akt/GSK-3 pathway in human colon cancer cells

The intestinal mucosa is a rapidly-renewing tissue characterized by cell proliferation, differentiation, and eventual apoptosis with progression up the vertical gut axis. The inhibition of phosphatidylinositol (PI) 3-kinase by specific chemical inhibitors or overexpression of the lipid phosphatase PTEN enhances enterocyte-like differentiation in human colon cancer cell models of intestinal differentiation. In this report, we examined the role of PI 3-kinase inhibition in the regulation of apoptotic gene expression in human colon cancer cell lines HT29, HCT-116, and Caco-2. Inhibition of PI 3-kinase with the chemical inhibitor wortmannin increased TNF-related apoptosis-inducing ligand (TRAIL; Apo2) mRNA and protein expression. Similarly, overexpression of the tumor suppressor protein PTEN, an antagonist of PI 3-kinase signaling, resulted in the increased expression of TRAIL. Activation of PI 3-kinase by pretreatment with IGF-1, a gut trophic factor, markedly attenuated the induction of TRAIL by wortmannin. Moreover, overexpression of active Akt, a downstream target of PI 3-kinase, or inhibition of GSK-3, a downstream target of active Akt, completely blocked the induction of TRAIL by wortmannin. Consistent with findings that TRAIL is induced by agents that enhance intestinal cell differentiation, TRAIL expression was specifically localized to the differentiated cells of the colon and small bowel. Adenovirus-mediated overexpression of TRAIL increased DNA fragmentation of HCT-116 cells, demonstrating the functional activity of TRAIL induction. Taken together, our findings demonstrate induction of the TRAIL by inhibition of PI 3-kinase in colon cancer cell lines. These results identify TRAIL, a novel TNF family member, as a downstream target of the PI 3-kinase/Akt/GSK-3 pathway and may have important implications for better understanding the role of the PI 3-kinase pathway in intestinal cell homeostasis.     

Presence of α-smooth muscle actin-positive endothelial cells in the luminal surface of adult aorta

α-Smooth muscle actin-positive endothelial cells have not been found in adult aortic endothelium except valve leaflets. Here, using en face immunostaining method, we identified α-smooth muscle actin-positive endothelial cells in the luminal surface of rat, mouse and human thoracic aortas. These cells express both endothelial markers and definite smooth muscle cell markers and were only occasionally observed in thoracic aorta of wild type mice and rats. Their density did not increase with aging. Given that α-smooth muscle actin-positive endothelial cells express low level of vascular endothelial-cadherin that is important for the maintenance of cell contact, these cells were frequently detected in the thoracic aorta of 5-week-old apolipoprotein-E deficient mice. In 20- to 24-week-old apolipoprotein-E deficient mice, marked accumulation of α-smooth muscle actin-positive endothelial cells was observed especially in the luminal surface of atheromatous plaques. Our findings indicate the existence of α-smooth muscle actin-positive endothelial cells in adult aortic endothelium and the possible association with progression of atherosclerosis.     

Regional functional heterogeneity of aortic smooth muscle cells in rats

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel’s wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells’ response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained.     

Activation dynamics and signaling properties of Notch3 receptor in the developing pulmonary artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.