Vascular Remodeling In Response Research Articles

Fat, fire and muscle – The role of adiponectin in pulmonary vascular inflammation and remodeling

Pulmonary hypertension is a life-threatening condition that results from a heterogeneous group of diseases, many of which demonstrate characteristic pathologic changes of pulmonary vascular inflammation and remodeling. Recent clinical studies indicate obesity to be a risk factor for the development of pulmonary hypertension; however, the mechanisms leading to this association are unknown. Adipocytes secrete multiple bioactive mediators that can influence inflammation and tissue remodeling, suggesting that adipose tissue may directly influence the pathogenesis of pulmonary hypertension. One of these mediators is adiponectin, a protein with a wide range of metabolic, anti-inflammatory, and anti-proliferative activities. Paradoxically, adiponectin is present in high concentration in the serum of lean healthy individuals, but decreases in obesity. Studies suggest that relative adiponectin-deficiency may contribute to the development of inflammatory diseases in obesity, and recent animal studies implicate adiponectin in the pathogenesis of pulmonary hypertension. Most notably, experimental studies show that adiponectin can reduce lung vascular remodeling in response to inflammation and hypoxia. Moreover, mice deficient in adiponectin develop a spontaneous lung vascular phenotype characterized by age-dependent increases in peri-vascular inflammatory cells and elevated pulmonary artery pressures. Emerging evidence indicates adiponectin's effects are mediated through anti-inflammatory and anti-proliferative actions on cells in the lung. This review aims to synthesize the existing data related to adiponectin's effects on the pulmonary vasculature and to discuss how changes in adiponectin levels might contribute to the development of pulmonary hypertension.

Platelet-Derived Growth Factor-BB Mediates Cell Migration through Induction of Activating Transcription Factor 4 and Tenascin-C

The acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-β. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration.

Macrophage LRP1 Suppresses Neo-Intima Formation during Vascular Remodeling by Modulating the TGF-β Signaling Pathway

BackgroundVascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated.Methodology/Principal FindingsTo define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-β2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-β signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-β2 and macrophages lacking LRP1 accumulate twice as much TGF-β2 in conditioned media. Finally, TNF-α modulation of the TGF-β2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-β2 in macrophages.Conclusions/SignificanceOur data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-β2 protein levels and to attenuate expression of the TGF-β2 gene resulting in suppression of the TGF-β signaling pathway.

What happens if you pose the wrong questions?

What happens if you pose the wrong questions?

A novel murine model of severe pulmonary arterial hypertension.

The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-β/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

C-type natriuretic peptide does not attenuate the development of pulmonary hypertension caused by hypoxia and VEGF receptor blockade

Aims C-type natriuretic peptide (CNP) is a local regulator of vascular tone and remodeling in many vascular beds. However, the role of CNP in modulating pulmonary arterial hypertensive and vascular remodeling responses is unclear. The purpose of this study was to determine if CNP is capable of preventing the development of pulmonary hypertension (PH). Main methods We used animal models of PH caused by chronic hypoxia alone or in combination with the vascular endothelial growth factor (VEGF) receptor blocker SU5416. We measured pulmonary hemodynamics, right ventricular hypertrophy and vascular remodeling effects in response to a continuous infusion of low dose or high dose CNP or vehicle placebo. Key findings Right ventricular hypertrophy and a marked elevation in right ventricular systolic pressure (RVSP) were seen in both models of PH. Rats treated with the combination of SU5416 and chronic hypoxia also developed pulmonary endothelial hyperproliferative lesions. Continuous intravenous infusion of CNP at either dose did not attenuate the development of PH, right ventricular hypertrophy or vascular remodeling in either of the models of PH despite a three-fold increase in serum CNP levels. Significance CNP does not prevent the development of PH in the chronic hypoxia or SU5416 plus hypoxia models of pulmonary hypertension suggesting that CNP may not play an important modulatory role in human PH.

Use of tensorial description in tissue remodeling: examples of F-actin distributions in pulmonary arteries in hypoxic hypertension.

A molecular configuration tensor Pij was introduced to analyze the distribution of fibrous proteins in vascular cells for studying cells and tissues biomechanics. We have used this technique to study the biomechanics of vascular remodeling in response to the changes of blood pressure and flow. In this paper, the remodeling of the geometrical arrangement of F-actin fibers in the smooth muscle cells in rat's pulmonary arteries in hypoxic hypertension was studied. The rats were exposed to a hypoxia condition of 10% for 0, 2, 12, and 24 hr at sea level. Remodeling of blood vessels were studied at the in vivo state under normal perfusion, no-load state when small rings from blood vessels were excised, and zero-stress state after the rings were cut open radially to release the residual stress. Tissue remodeling in response to changes in blood pressure is reflected in the zero-stress state. The tensor components were determined by analyzing the configuration of phalloidin stained F-actin fibers in the media layer of pulmonary arteries. The values of P31, P32, P33 in the in-vivo state, the no-load state, and the zero-stress state are obtained. This study demonstrated the distributions of fibrous molecules in tissue remodeling can be described quantitatively using the molecular configuration tensor.

Mechanisms of Cardiovascular Remodeling in Hyperhomocysteinemia

In hypertension, an increase in arterial wall thickness and loss of elasticity over time result in an increase in pulse wave velocity, a direct measure of arterial stiffness. This change is reflected in gradual fragmentation and loss of elastin fibers and accumulation of stiffer collagen fibers in the media that occurs independently of atherosclerosis. Similar results are seen with an elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), which increases vascular thickness, elastin fragmentation, and arterial blood pressure. Studies from our laboratory have demonstrated a decrease in elasticity and an increase in pulse wave velocity in HHcy cystathionine β synthase heterozygote knockout (CBS(-/+)) mice. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-TIMP (tissue inhibitor of metalloproteinase) inhibitory tertiary complex. We have demonstrated the contribution of the NO synthase (NOS) isoforms, endothelial NOS and inducible NOS, in the activation of latent MMP. The differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP resulting in vascular remodeling in response to HHcy. The contribution of the NOS isoforms, endothelial and inducible in the collagen/elastin switch, has been demonstrated. We have showed that an increase in inducible NOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance. In addition, increased levels of Hcy compete and suppress the γ-amino butyric acid-receptor, N-methyl-d-aspartate-receptor, and peroxisome proliferator-activated receptor. The HHcy causes oxidative stress by generating nitrotyrosine, activating the latent MMPs and decreasing the endothelial NO concentration. The HHcy causes elastinolysis and decrease elastic complicance of the vessel wall. The treatment with γ-amino butyric acid-receptor agonist (muscimol), N-methyl-d-aspartate-receptor antagonist (MK-801), and peroxisome proliferator-activated receptor agonists (ciprofibrate and ciglitazone) mitigates the cardiovascular dysfunction in HHcy [corrected].

Contribution of ASIC1 to development of right ventricular hypertrophy and pulmonary hypertension following chronic hypoxia

Chronic hypoxia (CH) associated with respiratory disease often results in pulmonary arterial constriction and vascular remodeling, ultimately leading to pulmonary hypertension and right heart failure. Several studies show increased vascular smooth muscle Ca2+ influx mediates both the vasoconstrictor and vascular remodeling responses to CH. Our laboratory has recently demonstrated a role for acid-sensing ion channel 1 (ASIC1) in mediating Ca2+ entry in pulmonary vascular smooth muscle. In addition, pulmonary vascular ASIC1 protein is elevated following CH. Therefore, we hypothesize ASIC1 contributes to the development of pulmonary hypertension. To test this hypothesis we examined 1) right ventricular systolic pressure (RVSP) as an indicator of pulmonary arterial pressure, 2) right ventricle to total heart weight (RV/T) and RV to body weight (RV/BW) ratios to assess right ventricular hypertrophy, and 3) hematocrit to determine the degree of polycythemia in control and CH (4wk @ 0.5 atm) ASIC1+/+ and ASIC1−/− mice. As expected, CH increased these variables in both ASIC1+/+ and ASIC1−/− mice, however ASIC1−/− CH mice displayed decreased RVSP, diminished RV/T and RV/BW ratio, and decreased hematocrit compared to ASIC1+/+ CH mice. These data suggest that ASIC1 plays a significant role in the development of CH-induced right ventricular hypertrophy and pulmonary hypertension.

Sustained release formulations of rhVEGF 165 produce a durable response in a murine model of peripheral angiogenesis

Local delivery of therapeutic angiogenic agents that stimulate blood vessel formation represents a promising strategy for the treatment of peripheral vascular disease (PVD). At present, requirements for temporal and spatial parameters for localized delivery are unclear, with a variety of sustained delivery approaches being examined. Two polymer-based sustained formulations containing the 165 amino acid isoform of human recombinant vascular endothelial growth factor-A (rhVEGF 165) were evaluated for their potential application in the treatment of PVD following intramuscular injection. Microspheres prepared from a 50:50 ratio of polylactic-co-glycolic acid (PLGA) and a gel of PLGA polymer solubilized in N-methyl pyrrolidone (PLGA:NMP) were each loaded with rhVEGF 165 and tested in vitro and in vivo. PLGA microspheres averaged ∼30 μm in diameter and contained 8.9% ( w/ w) rhVEGF 165, while the PLGA:NMP gel was formulated with varying amounts of spray freeze-dried rhVEGF 165 to result in final gel formulations having concentrations of 0.36, 0.72, or 3.6 mg/mL rhVEGF 165. In vitro release of rhVEGF 165 from PLGA microspheres showed ∼10% cumulative release by day 6, whereas the cumulative release of rhVEGF 165 from the PLGA:NMP gel matrices (0.65% w/ w loading) was less than 0.25% at this same time point. While the in vitro release characteristics of these two sustained release formulations were broadly different, the plasma rhVEGF 165 concentration–time profiles following hind-limb intramuscular (IM) injection of these formulations in non-compromised rats revealed similar in vivo pharmacokinetics. Three-dimensional resin casts of vascular architecture were prepared at days 3, 7, 14, 21, 28, 60, and 75 following a single IM dosing of these sustained release microsphere and gel matrix formulations in the gastrocnemius muscle of immune-compromised mice. Scanning electron microscopic visualization of these vascular casts demonstrated spatial arrangement of capillary sprouts and vessel enlargement consistent with profound vascular changes occurring within 3 days of dosing that persisted for 2 months, approximately 1 month beyond the anticipated completion of rhVEGF 165 release from these sustained delivery formulations. Vascular re-modeling events were correlated with histological and immunohistochemical parameters attributed to known biological actions of rhVEGF 165 signaling. Together, these pharmacokinetic and pharmacodynamic results support the use of sustained release PLGA-based formulations for the local delivery of rhVEGF 165 to achieve a durable vascular re-modeling response.

Blocking potassium channels: a new principle for treating restenosis?

This editorial refers to ‘Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers’ by A. Cheong et al ., pp. 282–289, this issue. Vascular smooth muscle cells are important for the structural integrity of the tunica media, but they are also central to vascular remodelling in response to injury. Although they are highly differentiated cells with the ability to regulate vascular tone and synthesize extracellular matrix, smooth muscle cells are also very plastic cells. This enables them to switch from a contractile to an invasive, migrating, and proliferating phenotype in response to local injury. Ion channels in the plasmalemma of smooth muscle cells have been implicated in this active process. There is evidence of profound changes in the types of ion channels that are expressed or functionally important during vascular smooth muscle cell transition resulting in occlusive vascular pathologies. In the systemic circulation, vessel restenosis remains a major factor limiting the success of balloon angioplasty/stenting of coronary, carotid, and femoral arteries.1 A critical event seems to be replacement of the KCa1.1 (BKCa) with the intermediate-conductance Ca2+-activated potassium channel KCa3.12 and the loss of Cav1.2 (the L-type voltage-dependent) calcium channels.3 This event confers functional dominance on other types of calcium channels, including the channel components transient receptor potential (TRP) C(canonical)1, …

Synergistic Therapeutic Effects of 2-Methoxyestradiol With Either Sildenafil or Bosentan on Amelioration of Monocrotaline-induced Pulmonary Hypertension and Vascular Remodeling

2-Methoxyestradiol (2ME) is a major nonestrogenic metabolite of estradiol. Our previous studies suggest that 2ME, in several models of cardiac and/or vascular injury, strongly inhibits cardiac and vascular remodeling. Furthermore, our most recent study shows that in male rats, 2ME attenuates the development and retards the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH), and in female rats, 2ME eliminates the exacerbation of PAH and the increased mortality due to ovariectomy. The current standard of care of patients with PAH includes treatment with an endothelin receptor antagonist (eg, bosentan) or a phosphodiesterase5 inhibitor (eg, sildenafil). Moreover, combination therapy is often prescribed. Therefore, in the present study, we compared the efficacy of 2ME (10 μg · kg(-1) · h(-1), 2ME-10) to the effects of bosentan (200 mg/kg; BOS), sildenafil (50 mg/kg; SIL), and their respective combinations with 2ME-10 (2ME + BOS and 2ME + SIL groups, respectively). Treatments were initiated 12 days after administration of MCT (60 mg/kg). Twenty-eight days after MCT administration, right ventricular peak systolic pressure was measured and morphometric analysis was conducted. 2ME exhibited beneficial effects in pulmonary hypertensive animals and had efficacy comparable to that of BOS and SIL. Importantly, combination treatments had favorable effects on survival, vascular remodeling, and inflammatory response, and the 2ME + SIL combination was significantly more efficacious than any other treatment. These results indicate that 2ME is effective in experimental PAH and suggests that 2ME may provide additional therapeutic benefit over existing drugs used for the treatment of pulmonary hypertension.

Decreased Neprilysin and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease

Studies with genetically engineered mice showed that decreased expression of the transmembrane peptidase neprilysin (NEP) increases susceptibility to hypoxic pulmonary vascular remodeling and hypertension; in hypoxic wild-type mice, expression is decreased early in distal pulmonary arteries, where prominent vascular remodeling occurs. Therefore, in humans with smoke- and hypoxia-induced vascular remodeling, as in chronic obstructive pulmonary disease (COPD), pulmonary activity/expression of NEP may likewise be decreased. To test whether NEP activity and expression are reduced in COPD lungs and pulmonary arterial smooth muscle cells (SMCs) exposed to cigarette smoke extract or hypoxia and begin to investigate mechanisms involved. Control and advanced COPD lung lysates (n = 13-14) were analyzed for NEP activity and protein and mRNA expression. As a control, dipeptidyl peptidase IV activity was analyzed. Lung sections were assessed for vascular remodeling and oxidant damage. Human pulmonary arterial SMCs were exposed to cigarette smoke extract, hypoxia, or H₂O₂, and incubated with antioxidants or lysosomal/proteasomal inhibitors. COPD lungs demonstrated areas of vascular rarification, distal muscularization, and variable intimal and prominent medial/adventitial thickening. NEP activity was reduced by 76%; NEP protein expression was decreased in alveolar walls and distal vessels; mRNA expression was also decreased. In SMCs exposed to cigarette smoke extract, hypoxia, and H₂O₂, NEP activity and expression were also reduced. Reactive oxygen species inactivated NEP activity; NEP protein degradation appeared to be substantially induced. Mechanisms responsible for reduced NEP activity and protein expression include oxidative reactions and protein degradation. Maintaining or increasing lung NEP may protect against pulmonary vascular remodeling in response to chronic smoke and hypoxia.

Redox Pioneer: Professor Gregg L. Semenza

Dr. Gregg L. Semenza is recognized here as a Redox Pioneer because he has published three articles that have been cited more than 1,000 times, and 74 articles that have each been cited more than 100 times. Dr. Semenza is known for his seminal discovery of hypoxia-inducible factor-1 (HIF-1) and for identifying molecular mechanisms underlying transcriptional responses to hypoxia. The discovery of HIF-1 is regarded as a fundamental breakthrough in understanding the molecular basis of O(2) sensing, and led to the identification of related transcriptional activators now known as the HIF family, which are the focus of many investigator's studies around the world. Understanding the molecular basis of cellular responses to hypoxia is of immense physiological significance and has tremendous implications for medicine. Compelling evidence now exists for the involvement of HIF-1 signaling in the progression of cancer, cardiovascular morbidities associated with aging and diabetes, and pulmonary vascular remodeling in response to chronic hypoxia. Dr. Semenza's findings have led to the development of inhibitors of the HIF-1 signaling pathway, which are currently being tested for treatment of cancer. He has received several honors for his scientific contributions, including the 2010 Gairdner Foundation International Award and election to the National Academy of Sciences of the United States of America.

PPARγ regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-κB

NADPH oxidases are a major source of superoxide production in the vasculature. The constitutively active Nox4 subunit, which is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension, is highly expressed in vascular wall cells. We demonstrated that rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), attenuated hypoxia-induced pulmonary hypertension, vascular remodeling, Nox4 induction, and reactive oxygen species generation in the mouse lung. The current study examined the molecular mechanisms involved in PPARγ-regulated, hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposing HPASMC to 1% oxygen for 72 h increased Nox4 gene expression and H(2)O(2) production, both of which were reduced by treatment with rosiglitazone during the last 24 h of hypoxia exposure or by treatment with small interfering RNA (siRNA) to Nox4. Hypoxia also increased HPASMC proliferation as well as the activity of a Nox4 promoter luciferase reporter, and these increases were attenuated by rosiglitazone. Chromatin immunoprecipitation assays demonstrated that hypoxia increased binding of the NF-κB subunit, p65, to the Nox4 promoter and that binding was attenuated by rosiglitazone treatment. The role of NF-κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity, whereas siRNA to p50 or p65 attenuated hypoxic stimulation of Nox4 promoter activity. These results provide novel evidence for NF-κB-mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ. These data provide new insights into potential mechanisms by which PPARγ activation inhibits Nox4 upregulation and the proliferation of cells in the pulmonary vascular wall to ameliorate pulmonary hypertension and vascular remodeling in response to hypoxia.

Functional consequences of the collagen/elastin switch in vascular remodeling in hyperhomocysteinemic wild-type, eNOS−/−, and iNOS−/− mice

A decrease in vascular elasticity and an increase in pulse wave velocity in hyperhomocysteinemic (HHcy) cystathionine-beta-synthase heterozygote knockout (CBS(-/+)) mice has been observed. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-tissue inhibitor of metalloproteinase (TIMP) inhibitory tertiary complex. However, the contribution of the nitric oxide synthase (NOS) isoforms eNOS and iNOS in the activation of latent MMP is unclear. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP, resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the NOS isoforms, endothelial and inducible, in the collagen/elastin switch. Experiments were performed on six groups of animals [wild-type (WT), eNOS(-/-), and iNOS(-/-) with and without homocysteine (Hcy) treatment (0.67 g/l) for 8-12 wk]. In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with trichrome and van Gieson stains, respectively, was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP-2 and -9, and elastin were measured by quantitative PCR and Western blot analyses. The 2% gelatin zymography was used to assess MMP activity. The increase in O(2)(-) and robust activity of MMP-9 in eNOS(-/-), WT+Hcy, and eNOS(-/-)+Hcy was accompanied by the gross disorganization and thickening of the ECM along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that an increase in iNOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance.

HIF-1 inhibition decreases systemic vascular remodelling diseases by promoting apoptosis through a hexokinase 2-dependent mechanism

Vascular remodelling diseases are characterized by the presence of proliferative and apoptosis-resistant vascular smooth muscle cells (VSMC). There is evidence that pro-proliferative and anti-apoptotic states are characterized by metabolic remodelling (a glycolytic phenotype with hyperpolarized mitochondria) involving Akt pathway activation by circulating growth factors. Hypoxia-inducible factor-1 (HIF-1) is involved in different vascular diseases. Since this transcription factor is implicated in metabolic responses, we hypothesized that HIF-1 activity could be involved in vascular remodelling in response to arterial injury. Our findings indicate that growth factors, such as platelet-derived growth factor (PDGF), activate the Akt pathway (measured by immunoblot) in human carotid artery VSMC. Activation of this pathway increased HIF-1 activation (measured by immunoblot), leading to increased glycolysis in VSMC. Expression and mitochondrial activity of hexokinase 2 (HXK2), a primary initiator of glycolysis, are increased during HIF-1 activation. The mitochondrial activity of HXK2 in VSMC led to the hyperpolarization of mitochondrial membrane potential (measured by tetramethylrhodamine methyl-ester perchlorate) and the suppression of apoptosis (measured by TUNEL assay and 3 activity), effects that are blocked by HIF-1 inhibition. Additionally, HIF-1 inhibition also decreased VSMC proliferation (proliferating cell nuclear antigen and Ki-67 assays). In vivo, we demonstrate that localized HIF-1 inhibition, using a dominant-negative HIF-1α adenoviral construct, prevented carotid artery post-injury remodelling in rats. We propose that HIF-1 is centrally involved in carotid artery remodelling in response to arterial injury and that localized inhibition of HIF-1 may be a novel therapeutic strategy to prevent carotid stenosis.

Molecular Pathways Involved in RAGE Mediated Attenuation of Arteriogenesis

The receptor for advanced glycation endproducts (RAGE) has been implicated in promoting vascular dysfunction through the activation of proinflammatory cascades and the induction of oxidative stress. Chronic activation of RAGE is associated with diabetes‐related vascular complications including coronary and peripheral vascular atherosclerosis and diabetic retinopathy. We have demonstrated that RAGE activation in monocytes and macrophages also regulates vascular remodeling in response to acute ischemia in a mouse model of hind‐limb ischemia. Lentiviral‐directed overexpression of RAGE in monocytes prevents the development of collateral vessels (arteriogenesis) in response to hind‐limb ischemia. To better understand the molecular mechanisms responsible for these observations, we have undertaken a cell culture approach to address the effect of monocyte ‐specific RAGE activation on the production of proarteriogenic and proangiogenic factors under hypoxic conditions and determine how chronic RAGE activation influences receptor recycling. We have generated soluble RAGE using a baculovirus system, which will provide a valuable reagent to evaluate RAGE signaling in response to hypoxic conditions. In addition, we are generating N‐terminal GFP‐tagged versions of the RAGE receptor that will allow us to evaluate RAGE receptor recycling following activation in a macrophage cell line.

Shear Stress Mediates Endothelial Adaptations to Exercise Training in Humans

Although episodic changes in shear stress have been proposed as the mechanism responsible for the effects of exercise training on the vasculature, this hypothesis has not been directly addressed in humans. We examined brachial artery flow-mediated dilation, an index of NO-mediated endothelial function, in healthy men in response to an acute bout of handgrip exercise and across an 8-week period of bilateral handgrip training. Shear stress responses were attenuated in one arm by cuff inflation to 60 mm Hg. Similar increases were observed in grip strength and forearm volume and girth in both limbs. Acute bouts of handgrip exercise increased shear rate (P<0.005) and flow-mediated dilation percentage (P<0.05) in the uncuffed limb, whereas no changes were evident in the cuffed arm. Handgrip training increased flow-mediated dilation percentage in the noncuffed limb at weeks 2, 4, and 6 (P<0.001), whereas no changes were observed in the cuffed arm. Brachial artery peak reactive hyperemia, an index of resistance artery remodeling, progressively increased with training in the noncuffed limb (P<0.001 and 0.004); no changes were evident in the cuffed arm. Neither acute nor chronic shear manipulation during exercise influenced endothelium-independent glyceryl trinitrate responses. These results demonstrate that exercise-induced changes in shear provide the principal physiological stimulus to adaptation in flow-mediated endothelial function and vascular remodeling in response to exercise training in healthy humans.