O-Linked ß-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

OBJECTIVE: Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked ß-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. APPROACH AND RESULTS: In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. CONCLUSIONS: These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat.

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

Estrogen effects on vascular inflammation are age dependent: role of estrogen receptors.

OBJECTIVE: 17ß-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent. APPROACH AND RESULTS: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERß. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice. CONCLUSIONS: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women.

Targeted delivery of pulmonary arterial endothelial cells overexpressing interleukin-8 receptors attenuates monocrotaline-induced pulmonary vascular remodeling.

OBJECTIVE: Interleukin-8 (IL-8) receptors IL8RA and IL8RB (IL8RA/B) on neutrophil membranes bind to IL-8 with high affinity and play a critical role in neutrophil recruitment to sites of injury and inflammation. This study tested the hypothesis that administration of rat pulmonary arterial endothelial cells (ECs) overexpressing IL8RA/B can accelerate the adhesion of ECs to the injured lung and inhibit monocrotaline-induced pulmonary inflammation, arterial thickening and hypertension, and right ventricular hypertrophy. APPROACH AND RESULTS: The treatment groups included 10-week-old ovariectomized Sprague-Dawley rats that received subcutaneous injection of PBS (vehicle), a single injection of monocrotaline (monocrotaline alone, 60 mg/kg, SC), monocrotaline followed by intravenous transfusion of ECs transduced with the empty adenoviral vector (null-EC), and monocrotaline followed by intravenous transfusion of ECs overexpressing IL8RA/B (1.5 × 10(6) cells/rat). Two days or 4 weeks after monocrotaline treatment, endothelial nitric oxide synthase, inducible nitric oxide synthase, cytokine-induced neutrophil chemoattractant-2ß (IL-8 equivalent in rat), and monocyte chemoattractant protein-1 expression, neutrophil and macrophage infiltration into pulmonary arterioles, and arteriolar and alveolar morphology were measured by histological and immunohistochemical techniques. Proinflammatory cytokine/chemokine protein levels were measured by Multiplex rat-specific magnetic bead-based sandwich immunoassay in total lung homogenates. Transfusion of ECs overexpressing IL8RA/B significantly reduced monocrotaline-induced neutrophil infiltration and proinflammatory mediator (IL-8, monocyte chemoattractant protein-1, inducible nitric oxide synthase, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2) expression in lungs and pulmonary arterioles and alveoli, pulmonary arterial pressure, and pulmonary arterial and right ventricular hypertrophy and remodeling. CONCLUSIONS: These provocative findings suggest that targeted delivery of ECs overexpressing IL8RA/B is effective in repairing the injured pulmonary vasculature.

Inhibiting C-reactive protein for the treatment of cardiovascular disease: promising evidence from rodent models.

Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.

Endothelial cells overexpressing interleukin-8 receptors reduce inflammatory and neointimal responses to arterial injury.

BACKGROUND: Interleukin-8 (IL8) receptors IL8RA and IL8RB on neutrophil membranes bind to IL8 and direct neutrophil recruitment to sites of inflammation, including acutely injured arteries. This study tested whether administration of IL8RA- and/or IL8RB-transduced rat aortic endothelial cells (ECs) accelerates adhesion of ECs to the injured surface, thus suppressing inflammation and neointima formation in balloon-injured rat carotid arteries. We tested the hypothesis that targeted delivery of ECs by overexpressing IL8RA and IL8RB receptors prevents inflammatory responses and promotes structural recovery of arteries after endoluminal injury. METHODS AND RESULTS: Young adult male rats received balloon injury of the right carotid artery and were transfused intravenously with ECs (total, 1.5×10(6) cells at 1, 3, and 5 hours after injury) transduced with adenoviral vectors carrying IL8RA, IL8RB, and IL8RA/RB (dual transduction) genes, AdNull (empty vector), or vehicle (no EC transfusion). ECs overexpressing IL8Rs inhibited proinflammatory mediators expression significantly (by 60% to 85%) and reduced infiltration of neutrophils and monocytes/macrophages into injured arteries at 1 day after injury, as well as stimulating a 2-fold increase in reendothelialization at 14 days after injury. IL8RA-EC, IL8RB-EC, and IL8RA/RB-EC treatment reduced neointima formation dramatically (by 80%, 74%, and 95%) at 28 days after injury. CONCLUSIONS: ECs with overexpression of IL8RA and/or IL8RB mimic the behavior of neutrophils that target and adhere to injured tissues, preventing inflammation and neointima formation. Targeted delivery of ECs to arteries with endoluminal injury provides a novel strategy for the prevention and treatment of cardiovascular disease.

Endothelial cells overexpressing IL-8 receptor reduce cardiac remodeling and dysfunction following myocardial infarction.

The endothelium is a dynamic component of the cardiovascular system that plays an important role in health and disease. This study tested the hypothesis that targeted delivery of endothelial cells (ECs) overexpressing neutrophil membrane IL-8 receptors IL8RA and IL8RB reduces acute myocardial infarction (MI)-induced left ventricular (LV) remodeling and dysfunction and increases neovascularization in the area at risk surrounding the infarcted tissue. MI was created by ligating the left anterior descending coronary artery in 12-wk-old male Sprague-Dawley rats. Four groups of rats were studied: group 1: sham-operated rats without MI or EC transfusion; group 2: MI rats with intravenous vehicle; group 3: MI rats with transfused ECs transduced with empty adenoviral vector (Null-EC); and group 4: MI rats with transfused ECs overexpressing IL8RA/RB (1.5 × 106 cells post-MI). Two weeks after MI, LV function was assessed by echocardiography; infarct size was assessed by triphenyltetrazolium chloride (live tissue) and picrosirus red (collagen) staining, and capillary density and neutrophil infiltration in the area at risk were measured by CD31 and MPO immunohistochemical staining, respectively. When compared with the MI + vehicle and MI-Null-EC groups, transfusion of IL8RA/RB-ECs decreased neutrophil infiltration and pro-inflammatory cytokine expression and increased capillary density in the area at risk, decreased infarct size, and reduced MI-induced LV dysfunction. These findings provide proof of principle that targeted delivery of ECs is effective in repairing injured cardiac tissue. Targeted delivery of ECs to infarcted hearts provides a potential novel strategy for the treatment of acute MI in humans.

Injury-activated transforming growth factor ß controls mobilization of mesenchymal stem cells for tissue remodeling.

Upon secretion, transforming growth factor ß (TGFß) is maintained in a sequestered state in extracellular matrix as a latent form. The latent TGFß is considered as a molecular sensor that releases active TGFß in response to the perturbations of the extracellular matrix at the situations of mechanical stress, wound repair, tissue injury, and inflammation. The biological implication of the temporal discontinuity of TGFß storage in the matrix and its activation is obscure. Here, using several animal models in which latent TGFß is activated in vascular matrix in response to injury of arteries, we show that active TGFß controls the mobilization and recruitment of mesenchymal stem cells (MSCs) to participate in tissue repair and remodeling. MSCs were mobilized into the peripheral blood in response to vascular injury and recruited to the injured sites where they gave rise to both endothelial cells for re-endothelialization and myofibroblastic cells to form thick neointima. TGFßs were activated in the vascular matrix in both rat and mouse models of mechanical injury of arteries. Importantly, the active TGFß released from the injured vessels is essential to induce the migration of MSCs, and cascade expression of monocyte chemotactic protein-1 stimulated by TGFß amplifies the signal for migration. Moreover, sustained high levels of active TGFß were observed in peripheral blood, and at the same time points following injury, Sca1+ CD29+ CD11b- CD45- MSCs, in which 91% are nestin+ cells, were mobilized to peripheral blood and recruited to the remodeling arteries. Intravenously injection of recombinant active TGFß1 in uninjured mice rapidly mobilized MSCs into circulation. Furthermore, inhibitor of TGFß type I receptor blocked the mobilization and recruitment of MSCs to the injured arteries. Thus, TGFß is an injury-activated messenger essential for the mobilization and recruitment of MSCs to participate in tissue repair/remodeling.

Transforming growth factor-ß regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure.

We have previously shown that inhibition of transforming growth factor-ß (TGF-ß) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-ß signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-ß neutralizing antibody), or vehicle. Lung function and development and TGF-ß and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-ß synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-ß signaling in hypoxia-exposed animals. Inhibition of TGF-ß signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-ß signaling indicates that TGF-ß is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.

Acute O-GlcNAcylation prevents inflammation-induced vascular dysfunction.

Acute increases in cellular protein O-linked N-acetyl-glucosamine (O-GlcNAc) modification (O-GlcNAcylation) have been shown to have protective effects in the heart and vasculature. We hypothesized that d-glucosamine (d-GlcN) and Thiamet-G, two agents that increase protein O-GlcNAcylation via different mechanisms, inhibit TNF-α-induced oxidative stress and vascular dysfunction by suppressing inducible nitric oxide (NO) synthase (iNOS) expression. Rat aortic rings were incubated for 3h at 37°C with d-GlcN or its osmotic control l-glucose (l-Glc) or with Thiamet-G or its vehicle control (H(2)O) followed by the addition of TNF-α or vehicle (H(2)O) for 21 h. After incubation, rings were mounted in a myograph to assess arterial reactivity. Twenty-four hours of incubation of aortic rings with TNF-α resulted in 1) a hypocontractility to 60 mM K(+) solution and phenylephrine, 2) blunted endothelium-dependent relaxation responses to ACh and substance P, and 3) unaltered relaxing response to the Ca(2+) ionophore A-23187 and the NO donor sodium nitroprusside compared with aortic rings cultured in the absence of TNF-α. d-GlcN and Thiamet-G pretreatment suppressed the TNF-α-induced hypocontractility and endothelial dysfunction. Total protein O-GlcNAc levels were significantly higher in aortic segments treated with d-GlcN or Thiamet-G compared with controls. Expression of iNOS protein was increased in TNF-α-treated rings, and this was attenuated by pretreatment with either d-GlcN or Thiamet-G. Dense immunostaining for nitrotyrosylated proteins was detected in the endothelium and media of the aortic wall, suggesting enhanced peroxynitrite production by iNOS. These findings demonstrate that acute increases in protein O-GlcNAcylation prevent TNF-α-induced vascular dysfunction, at least in part, via suppression of iNOS expression.

Hypoxia induces downregulation of PPAR-γ in isolated pulmonary arterial smooth muscle cells and in rat lung via transforming growth factor-ß signaling.

Chronic hypoxia activates transforming growth factor-ß (TGF-ß) signaling and leads to pulmonary vascular remodeling. Pharmacological activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) has been shown to prevent hypoxia-induced pulmonary hypertension and vascular remodeling in rodent models, suggesting a vasoprotective effect of PPAR-γ under chronic hypoxic stress. This study tested the hypothesis that there is a functional interaction between TGF-ß/Smad signaling pathway and PPAR-γ in isolated pulmonary artery small muscle cells (PASMCs) under hypoxic stress. We observed that chronic hypoxia led to a dramatic decrease of PPAR-γ protein expression in whole lung homogenates (rat and mouse) and hypertrophied pulmonary arteries and isolated PASMCs. Using a transgenic model of mouse with inducible overexpression of a dominant-negative mutant of TGF-ß receptor type II, we demonstrated that disruption of TGF-ß pathway significantly attenuated chronic hypoxia-induced downregulation of PPAR-γ in lung. Similarly, in isolated rat PASMCs, antagonism of TGF-ß signaling with either a neutralizing antibody to TGF-ß or the selective TGF-ß receptor type I inhibitor SB431542 effectively attenuated hypoxia-induced PPAR-γ downregulation. Furthermore, we have demonstrated that TGF-ß1 treatment suppressed PPAR-γ expression in PASMCs under normoxia condition. Chromatin immunoprecipitation analysis showed that TGF-ß1 treatment significantly increased binding of Smad2/3, Smad4, and the transcriptional corepressor histone deacetylase 1 to the PPAR-γ promoter in PASMCs. Conversely, treatment with the PPAR-γ agonist rosiglitazone attenuated TGF-ß1-induced extracellular matrix molecule expression and growth factor in PASMCs. These data provide strong evidence that activation of TGF-ß/Smad signaling, via transcriptional suppression of PPAR-γ expression, mediates chronic hypoxia-induced downregulation of PPAR-γ expression in lung.

Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone.

Hypoxia enhances transforming growth factor-ß (TGF-ß) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-ß signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-ß signaling, and collagen content were made. To determine whether excessive TGF-ß signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-ß-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-ß signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-ß signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-ß signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-ß signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-ß signaling and improving alveolar development.

Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy.

RATIONALE: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. OBJECTIVE: Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. METHODS AND RESULTS: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. CONCLUSIONS: These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.

C-reactive protein-mediated vascular injury requires complement.

BACKGROUND: We previously demonstrated that vascular injury-induced neointima formation is exaggerated in human C-reactive protein (CRP) transgenic (CRPtg) compared to nontransgenic (NTG) mice. We now test the hypothesis that complement is required for this effect. METHODS AND RESULTS: CRPtg and NTG with a normal complement system versus their counterparts lacking expression of complement component 3 (C3) protein (CRPtg/C3(-/-) and NTG/C3(-/-)) underwent carotid artery ligation. Twenty-eight days later, the injured vessels in CRPtg had thicker neointimas and more immunoreactive C3 in the surrounding adventitia compared with NTG. In CRPtg/C3(-/-), there was no increase in neointimal thickness compared with NTG or NTG/C3(-/-). Decreasing human CRP blood levels through administration of a selective antisense oligonucleotide eliminated the depletion of serum C3 associated with vascular injury and reduced immunoreactive C3 in the resultant lesions. In injured vessels, C3 colocalized with F4/80 (macrophage marker), and in vitro, human CRP elicited increased expression of C3 by bone marrow-derived macrophages. CONCLUSIONS: Human CRP exaggeration of neointima formation in injured mouse carotid arteries associates with decreased circulating C3 and increased tissue-localized C3. C3 elimination or pharmacological reduction of human CRP prevents CRP-driven exacerbation of the injury response. In the CRPtg model system, mouse C3 is essential for the effect of human CRP.

Inhibition of transforming growth factor-beta signaling induces left ventricular dilation and dysfunction in the pressure-overloaded heart.

This study utilized a transgenic mouse model that expresses an inducible dominant-negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII) to define the structural and functional responses of the left ventricle (LV) to pressure-overload stress in the absence of an intact TGF-beta signaling cascade. DnTGFbetaRII and nontransgenic (NTG) control mice (male, 8-10 wk) were randomized to receive Zn(2+) (25 mM ZnSO(4) in drinking H(2)O to induce DnTGFbetaRII gene expression) or control tap H(2)O and then further randomized to undergo transverse aortic constriction (TAC) or sham surgery. At 7 days post-TAC, interstitial nonmyocyte proliferation (Ki67 staining) was greatly reduced in LV of DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. At 28 and 120 days post-TAC, collagen deposition (picrosirius-red staining) in LV was attenuated in DnTGFbetaRII+Zn(2+) mice compared with the other TAC groups. LV end systolic diameter and end systolic and end diastolic volumes were markedly increased, while ejection fraction and fractional shortening were significantly decreased in TAC-DnTGFbetaRII+Zn(2+) mice compared with the other groups at 120 days post-TAC. These data indicate that interruption of TGF-beta signaling attenuates pressure-overload-induced interstitial nonmyocyte proliferation and collagen deposition and promotes LV dilation and dysfunction in the pressure-overloaded heart, thus creating a novel model of dilated cardiomyopathy.

Stainless steel ions stimulate increased thrombospondin-1-dependent TGF-beta activation by vascular smooth muscle cells: implications for in-stent restenosis.

BACKGROUND/AIMS: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-beta is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-beta antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-beta activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-beta activation and VSMC phenotype change. METHODS: VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-beta activity were assessed by immunoblotting. RESULTS: SS ions stimulate the synthetic phenotype, increased TGF-beta activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression. CONCLUSIONS: These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-beta activation.

Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts.

This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G signaling inhibits transforming growth factor (TGF)-beta1-induced extracellular matrix expression in cardiac fibroblasts and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Left ventricular hypertrophy and fibrosis, collagen deposition, and myofibroblast transformation of cardiac fibroblasts in response to pressure overload by transverse aortic constriction were exaggerated in ANP-null mice compared with wild-type controls. ANP and cGMP inhibited TGF-beta1-induced myofibroblast transformation, proliferation, collagen synthesis, and plasminogen activator inhibitor-1 expression in cardiac fibroblasts isolated from wild-type mice. Following pretreatment with cGMP, TGF-beta1 induced phosphorylation of Smad3, but the resultant pSmad3 could not be translocated to the nucleus. pSmad3 that had been phosphorylated with recombinant protein kinase G-1alpha was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry. The analysis revealed phosphorylation of Ser309 and Thr388 residues, sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-beta receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3. These results suggest that phosphorylation of Smad3 by protein kinase G is a potential molecular mechanism by which activation of ANP/cGMP/protein kinase G signaling disrupts TGF-beta1-induced nuclear translocation of pSmad3 and downstream events, including myofibroblast transformation, proliferation, and expression of extracellular matrix molecules in cardiac fibroblasts. We postulate that this process contributes to the antifibrogenic effects of the natriuretic peptide in heart.

Estrogen and mechanisms of vascular protection.

Estrogen has antiinflammatory and vasoprotective effects when administered to young women or experimental animals that appear to be converted to proinflammatory and vasotoxic effects in older subjects, particularly those that have been hormone free for long periods. Clinical studies have raised many important questions about the vascular effects of estrogen that cannot easily be answered in human subjects. Here we review cellular/molecular mechanisms by which estrogen modulates injury-induced inflammation, growth factor expression, and oxidative stress in arteries and isolated vascular smooth muscle cells, with emphasis on the role of estrogen receptors and the nuclear factor-kappaB (NFkappaB) signaling pathway, as well as evidence that these protective mechanisms are lost in aging subjects.

Apolipoprotein A-I mimetic peptide treatment inhibits inflammatory responses and improves survival in septic rats.

Systemic inflammation induces a multiple organ dysfunction syndrome that contributes to morbidity and mortality in septic patients. Since increasing plasma apolipoprotein A-I (apoA-I) and HDL may reduce the complications of sepsis, we tested the hypothesis that the apoA-I mimetic peptide 4F confers similar protective effects in rats undergoing cecal ligation and puncture (CLP) injury. Male Sprague-Dawley rats were randomized to undergo CLP or sham surgery. IL-6 levels were significantly elevated in plasma by 6 h after CLP surgery compared with shams. In subsequent studies, CLP rats were further subdivided to receive vehicle or 4F (10 mg/kg) by intraperitoneal injection, 6 h after sepsis induction. Sham-operated rats received saline. Echocardiographic studies showed a reduction in left ventricular end-diastolic volume, stroke volume, and cardiac output (CO) 24 h after CLP surgery. These changes were associated with reduced blood volume and left ventricular filling pressure. 4F treatment improved blood volume status, increased CO, and reduced plasma IL-6 in CLP rats. Total cholesterol (TC) and HDL were 79 +/- 5 and 61 +/- 4 mg/dl, respectively, in sham rats. TC was significantly reduced in CLP rats (54 +/- 3 mg/dl) due to a reduction in HDL (26 +/- 3 mg/dl). 4F administration to CLP rats attenuated the reduction in TC (69 +/- 4 mg/dl) and HDL (41 +/- 3 mg/dl) and prevented sepsis-induced changes in HDL protein composition. Increased plasma HDL in 4F-treated CLP rats was associated with an improvement in CO and reduced mortality. It is proposed that protective effects of 4F are related to its ability to prevent the sepsis-induced reduction in plasma HDL.

Induced pluripotent stem cell-derived endothelial cells attenuate lipopolysaccharide-induced acute lung injury.

The chemokine receptors CXCR1/2 and CCR2/5 play a critical role in neutrophil and monocyte recruitment to sites of injury and/or inflammation. Neutrophil-mediated inflammation and endothelial cell (EC) injury are unifying factors in the pathogenesis of the acute respiratory distress syndrome. This study tested the hypothesis that systemic administration of rat-induced pluripotent stem cell (iPS)-derived ECs (iPS-ECs) overexpressing CXCR1/2 or CCR2/5 attenuates lipopolysaccharide (LPS)-induced acute lung injury. Rat iPS-ECs were transduced with adenovirus containing cDNA of CXCR1/2 or CCR2/5. Ovariectomized Sprague-Dawley rats (10 wk old) received intraperitoneal injection of LPS and intravenous infusion of 1) saline vehicle, 2) AdNull-iPS-ECs (iPS-ECs transduced with empty adenoviral vector), 3) CXCR1/2-iPS-ECs (iPS-ECs overexpressing CXCR1/2), or 4) CCR2/5-iPS-ECs (iPS-ECs overexpressing CCR2/5) at 2 h post-LPS. Rats receiving intraperitoneal injection of saline served as sham controls. Later (4 h), proinflammatory cytokine/chemokine mRNA and protein levels were measured in total lung homogenates by real-time RT-PCR and Luminex multiplex assays, and neutrophil and macrophage infiltration in alveoli was measured by immunohistochemical staining. Pulmonary microvascular permeability was assessed by the Evans blue technique, and pulmonary edema was estimated by wet-to-dry lung weight ratios. Albumin levels and neutrophil counts were assessed in bronchoalveolar lavage fluid at 24 h post-LPS. Both CXCR1/2-iPS-ECs and CCR2/5-iPS-ECs significantly reduced LPS-induced proinflammatory mediator expression, neutrophil and macrophage infiltration, pulmonary edema, and vascular permeability compared with controls. These provocative findings provide strong evidence that targeted delivery of iPS-ECs overexpressing CXCR1/2 or CCR2/5 prevents LPS-induced acute lung injury.NEW & NOTEWORTHY We have developed a novel approach to address neutrophil-mediated inflammation and endothelial damage by targeted delivery of rat-induced pluripotent stem cell (iPS)-derived endothelial cell (ECs)overexpressing chemokine receptors CXCR1/2 and CCR2/5 in injured lung tissue in a model of acute lung injury. We have demonstrated that intravenously transfused CXCR1/2-iPS-ECs and CCR2/5-iPS-ECs are recruited to lipopolysaccharide-injured lungs and attenuate lipopolysaccharide-induced parenchymal lung injury responses, including inflammatory mediator expression, inflammatory cell infiltration, and vascular leakage compared with controls.