Knockdown of connective tissue growth factor by plasmid-based short hairpin RNA prevented pulmonary vascular remodeling in cigarette smoke-exposed rats

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. Connective tissue growth factor (CTGF) is a cysteine-rich peptide implicated in several biological processes such as cell proliferation, survival, and migration. This study investigated the potential role of CTGF in pulmonary vascular remodeling. We constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of CTGF in primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) and in rat lung vessels. Rat PASMCs were challenged with cigarette smoke extract (CSE). Rats were exposed to cigarette smoke for 3 months in the absence or in the presence of plasmid-based short hairpin RNA against CTGF which was administrated by tail vein injection. CTGFshRNA significantly prevented CTGF and cyclin D1 expression, arrested cell cycle at G0/G1 phase and suppressed cell proliferation in rPASMCs exposed to CSE. CTGFshRNA administration ameliorated pulmonary vascular remodeling, inhibited cigarette smoke-induced CTGF elevation and reversed the cyclin D1 increase in pulmonary vessels in rats. Collectively, our data demonstrated that plasmid-based shRNA against CTGF attenuated pulmonary vascular remodeling in cigarette smoke-exposed rats.     

Atrial natriuretic peptide in hypoxia

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. Among them, atrial natriuretic peptide (ANP) synthesis and secretion is increased during hypoxic exposure and plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases, including chronic hypoxia-induced pulmonary hypertension and vascular remodeling, and right ventricular hypertrophy and right heart failure. This review discusses the roles of ANP and its receptors in hypoxia-induced pulmonary hypertension. We and other investigators have demonstrated that ANP gene expression is enhanced by exposure to hypoxia and that the ANP so generated protects against the development of hypoxic pulmonary hypertension. Results also show that hypoxia directly stimulates ANP gene expression and ANP release in cardiac myocytes in vitro. Several cis-responsive elements of the ANP promoter are involved in the response to changes in oxygen tension. Further, the ANP clearance receptor NPR-C, but not the biological active NPR-A and NPR-B receptors, is downregulated in hypoxia adapted lung. Hypoxia-sensitive tyrosine kinase receptor-associated growth factors, including fibroblast growth factor (FGF) and platelet derived growth factor (PDGF)-BB, but not hypoxia per se, inhibit NPR-C gene expression in pulmonary arterial smooth muscle cells in vitro. The reductions in NPR-C in the hypoxic lung retard the clearance of ANP and allow more ANP to bind to biological active NPR-A and NPR-B in the pulmonary circulation, relaxing preconstricted pulmonary vessels, reducing pulmonary arterial pressure, and attenuating the development of hypoxia-induced pulmonary hypertension and vascular remodeling.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

Extracellular superoxide dismutase in vessels and airways of humans and baboons

Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO^·) species, is a primary mediator of vascular relaxation. During NO^·′ diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO^·. High levels of extracellualar superoxide dismutase in vessels could, therefore, be essential to enable NO' to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.     

Apelin/APJ：血管功能调节因子和药物治疗新靶点

Apelin(APJendogenousligand)是血管紧张素Ⅱ1型受体相关蛋白(angiotensin receptor-like 1,APJ)的内源性配体.Apelin/APJ系统在机体内广泛分布,在众多血管系统表达水平较高,如心血管系统、肺血管系统等.研究发现,apelin可调节血管张力,促进血管平滑肌细胞增殖、视网膜血管新生以及单核细胞向内皮细胞黏附,促进肝门静脉和冠状动脉侧枝形成等.本文就apelin调节血管功能及其相关疾病(高血压、肺动脉高压、动脉粥样硬化、胶质瘤、肺癌、门静脉高压、糖尿病血管并发症等)进行综述,揭示了apelin与血管及其相关疾病的内在联系,表明apelin/APJ可作为血管疾病的治疗靶点.     

The Multiscale Systems Immunology project: software for cell-based immunological simulation

Background  

Computer simulations are of increasing importance in modeling biological phenomena. Their purpose is to predict behavior and guide future experiments. The aim of this project is to model the early immune response to vaccination by an agent based immune response simulation that incorporates realistic biophysics and intracellular dynamics, and which is sufficiently flexible to accurately model the multi-scale nature and complexity of the immune system, while maintaining the high performance critical to scientific computing.     

Altered pulmonary vasoreactivity in the chronically hypoxic lung

Prolonged exposure to alveolar hypoxia induces physiological changes in the pulmonary vasculature that result in the development of pulmonary hypertension. A hallmark of hypoxic pulmonary hypertension is an increase in vasomotor tone. In vivo, pulmonary arterial smooth muscle cell contraction is influenced by vasoconstrictor and vasodilator factors secreted from the endothelium, lung parenchyma and in the circulation. During chronic hypoxia, production of vasoconstrictors such as endothelin-1 and angiotensin II is enhanced locally in the lung, while synthesis of vasodilators may be reduced. Altered reactivity to these vasoactive agonists is another physiological consequence of chronic exposure to hypoxia. Enhanced contraction in response to endothelin-1 and angiotensin II, as well as depressed vasodilation in response to endothelium-derived vasodilators, has been documented in models of hypoxic pulmonary hypertension. Chronic hypoxia may also have direct effects on pulmonary vascular smooth muscle cells, modulating receptor population, ion channel activity or signal transduction pathways. Following prolonged hypoxic exposure, pulmonary vascular smooth muscle exhibits alterations in K+ current, membrane depolarization, elevation in resting cytosolic calcium and changes in signal transduction pathways. These changes in the electrophysiological parameters of pulmonary vascular smooth muscle cells are likely associated with an increase in basal tone. Thus, hypoxia-induced modifications in pulmonary arterial myocyte function, changes in synthesis of vasoactive factors and altered vasoresponsiveness to these agents may shift the environment in the lung to one of contraction instead of relaxation, resulting in increased pulmonary vascular resistance and elevated pulmonary arterial pressure.     

Modeling and simulation of biological systems with stochasticity

Mathematical modeling is a powerful approach for understanding the complexity of biological systems. Recently, several successful attempts have been made for simulating complex biological processes like metabolic pathways, gene regulatory networks and cell signaling pathways. The pathway models have not only generated experimentally verifiable hypothesis but have also provided valuable insights into the behavior of complex biological systems. Many recent studies have confirmed the phenotypic variability of organisms to an inherent stochasticity that operates at a basal level of gene expression. Due to this reason, development of novel mathematical representations and simulations algorithms are critical for successful modeling efforts in biological systems. The key is to find a biologically relevant representation for each representation. Although mathematically rigorous and physically consistent, stochastic algorithms are computationally expensive, they have been successfully used to model probabilistic events in the cell. This paper offers an overview of various mathematical and computational approaches for modeling stochastic phenomena in cellular systems.     

A computational modeling and analysis in cell biological dynamics using electric cell-substrate impedance sensing (ECIS)

In this paper, a study of computational modeling and multi-scale analysis in cell dynamics is presented. Our study aims at: (1) deriving and validating a mathematical model for cell growth, and (2) quantitatively detecting and analyzing the biological interdependencies across multiple observational scales with a variety of time and frequency resolutions. This research was conducted using the time series data practically measured from a novel on-line cell monitoring technique, referred to as electric cell-substrate impedance sensing (ECIS), which allows continuously tracking the cellular behavior such as adhesion, proliferation, spreading and micromotion. First, comparing our ECIS-based cellular growth modeling analysis results with those determined by hematocytometer measurement using different time intervals, we found that the results obtained from both experimental methods consistently agreed. However, our study demonstrated that it is much easier and more convenient to operate with the ECIS system for on-line cellular growth monitoring. Secondly, for multi-scale analysis our results showed that the proposed wavelet-based methodology can effectively quantify the fluctuations associated with cell micromotions and quantitatively capture the biological interdependencies across multiple observational scales. Note that although the wavelet method is well known, its application into the ECIS time series analysis is novel and unprecedented in computational cell biology. Our analyses indicated that the proposed study on ECIS time series could provide a hopeful start and great potentials in both modeling and elucidating the complex mechanisms of cell biological systems.     

Notch Signaling Change in Pulmonary Vascular Remodeling in Rats with Pulmonary Hypertension and Its Implication for Therapeutic Intervention

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1–4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1–2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.     

Stratified Pulmonary Blood Flow: Some Consequences in Emphysema and Pulmonary Embolism

Both ventilation and blood flow in the secondary lobule of the lung are stratified; each unit of lung tissue in the proximal portion of the lobule receives up to four times the blood flow of units in the peripheral portion. Questions of the limiting role of gas diffusion within the small airways become virtually irrelevant in the face of this stratification of function.The central portion of the lobule, with its high ventilation, blood flow, and gas exchange, is very vulnerable; small lesions at this site will produce disproportionately large disturbances of gas exchange and of pulmonary vascular resistance. This may well account for some of the phenomena of conditions such as centrilobular emphysema and pulmonary microembolism.     

Intratracheal mesenchymal stem cell administration attenuates monocrotaline-induced pulmonary hypertension and endothelial dysfunction

The administration of mesenchymal stem cells (MSCs) has been proposed for the treatment of pulmonary hypertension. However, the effect of intratracheally administered MSCs on the pulmonary vascular bed in monocrotaline-treated rats has not been determined. In the present study, the effect of intratracheal administration of rat MSCs (rMSCs) on monocrotaline-induced pulmonary hypertension and impaired endothelium-dependent responses were investigated in the rat. Intravenous injection of monocrotaline increased pulmonary arterial pressure and vascular resistance and decreased pulmonary vascular responses to acetylcholine without altering responses to sodium nitroprusside and without altering systemic responses to the vasodilator agents when responses were evaluated at 5 wk. The intratracheal injection of 3 x 10(6) rMSCs 2 wk after administration of monocrotaline attenuated the rise in pulmonary arterial pressure and pulmonary vascular resistance and restored pulmonary responses to acetylcholine toward values measured in control rats. Treatment with rMSCs decreased the right ventricular hypertrophy induced by monocrotaline. Immunohistochemical studies showed widespread distribution of lacZ-labeled rMSCs in lung parenchyma surrounding airways in monocrotaline-treated rats. Immunofluorescence studies revealed that transplanted rMSCs retained expression of von Willebrand factor and smooth muscle actin markers specific for endothelial and smooth muscle phenotypes. However, immunolabeled cells were not detected in the wall of pulmonary vessels. These data suggest that the decrease in pulmonary vascular resistance and improvement in response to acetylcholine an endothelium-dependent vasodilator in monocrotaline-treated rats may result from a paracrine effect of the transplanted rMSCs in lung parenchyma, which improves vascular endothelial function in the monocrotaline-injured lung.     

The lung HETEs (and EETs) up

Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.     

Carbon monoxide promotes hypoxic pulmonary vascular remodeling

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.     

Multi-scale models of lung fibrosis

The architectural complexity of the lung is crucial to its ability to function as an organ of gas exchange; the branching tree structure of the airways transforms the tracheal cross-section of only a few square centimeters to a blood-gas barrier with a surface area of tens of square meters and a thickness on the order of a micron or less. Connective tissue comprised largely of collagen and elastic fibers provides structural integrity for this intricate and delicate system. Homeostatic maintenance of this connective tissue, via a balance between catabolic and anabolic enzyme-driven processes, is crucial to life. Accordingly, when homeostasis is disrupted by the excessive production of connective tissue, lung function deteriorates rapidly with grave consequences leading to chronic lung conditions such as pulmonary fibrosis. Understanding how pulmonary fibrosis develops and alters the link between lung structure and function is crucial for diagnosis, prognosis, and therapy. Further information gained could help elaborate how the healing process breaks down leading to chronic disease. Our understanding of fibrotic disease is greatly aided by the intersection of wet lab studies and mathematical and computational modeling. In the present review we will discuss how multi-scale modeling has facilitated our understanding of pulmonary fibrotic disease as well as identified opportunities that remain open and have produced techniques that can be incorporated into this field by borrowing approaches from multi-scale models of fibrosis beyond the lung.     

Pulmonary vascular dysfunction in preterm lambs with chronic lung disease

Chronic lung injury from prolonged mechanical ventilation after premature birth inhibits the normal postnatal decrease in pulmonary vascular resistance (PVR) and leads to structural abnormalities of the lung circulation in newborn sheep. Compared with normal lambs born at term, chronically ventilated preterm lambs have increased pulmonary arterial smooth muscle and elastin, fewer lung microvessels, and reduced abundance of endothelial nitric oxide synthase. These abnormalities may contribute to impaired respiratory gas exchange that often exists in infants with chronic lung disease (CLD). Nitric oxide inhalation (iNO) reduces PVR in human infants and lambs with persistent pulmonary hypertension. We wondered whether iNO might have a similar effect in lambs with CLD. We therefore studied the effect of iNO on PVR in lambs that were delivered prematurely at approximately 125 days of gestation (term = 147 days) and mechanically ventilated for 3 wk. All of the lambs had chronically implanted catheters for measurement of pulmonary vascular pressures and blood flow. During week 2 of mechanical ventilation, iNO at 15 parts/million for 1 h decreased PVR by approximately 20% in 12 lambs with evolving CLD. When the same study was repeated in eight lambs at the end of week 3, iNO had no significant effect on PVR. To see whether this loss of iNO effect on PVR might reflect dysfunction of lung vascular smooth muscle, we infused 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP; 150 microg. kg(-1). min(-1) iv) for 15-30 min in four of these lambs at the end of week 3. PVR consistently decreased by 30-35%. Lung immunohistochemistry and immunoblot analysis of excised pulmonary arteries from lambs with CLD, compared with control term lambs, showed decreased soluble guanylate cyclase (sGC). These results suggest that loss of pulmonary vascular responsiveness to iNO in preterm lambs with CLD results from impaired signaling, possibly related to deficient or defective activation of sGC, the intermediary enzyme through which iNO induces increased vascular smooth muscle cell cGMP and resultant vasodilation.     

Heparin-like molecules inhibit pulmonary vascular pericyte proliferation in vitro

Proliferation of vascular pericytes (PCs), smooth muscle-like cells found in the distal microvasculature, contributes to vascular remodeling in pulmonary hypertension. The factors controlling lung PC quiescence in normal states are poorly understood. We demonstrate that exogenous heparin and heparan sulfate proteoglycans inhibit rat lung PC proliferation in vitro as does pulmonary vascular subendothelial matrix, particularly its heparan sulfate component. Heparin inhibits the intracellular alkalinization essential to proliferation, and we show that inhibition of alkalinization by 5-(N, N-dimethyl)amiloride also reduces PC proliferation. As shown by DNA staining and fluorescence-activated cell sorting analysis, heparin does not induce apoptosis in PCs. However, heparin maintains lung PCs in the G(0)/G(1) growth phase. Heparin induces production of p21, a potent inhibitor of cyclin-dependent kinases, thereby potentially identifying a fundamental mechanism by which heparin inhibits proliferation in smooth muscle-like cells. These studies establish additional similarities between lung PCs and smooth muscle cells and provide further understanding of growth control in the lung microvasculature. They also further support the rationale that heparin-like molecules might be therapeutically beneficial in pulmonary hypertension.