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1.
Circ Res ; 133(11): 885-898, 2023 11 10.
Article in English | MEDLINE | ID: mdl-37929582

ABSTRACT

BACKGROUND: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are poorly understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. METHODS: Eight-week-old male and female C57BL/6J mice received either Nγ-nitro-L-arginine methyl ester and high-fat diet or control water and diet for 2, 5, and 12 weeks. The db/db mice were studied as a second model of HFpEF. Early pathways regulating PH were identified by bulk and single-cell RNA sequencing. Findings were confirmed by immunostain in lungs of mice or lung slides from clinically performed autopsies of patients with PH-HFpEF. ELISA was used to verify IL-1ß (interleukin-1 beta) in mouse lung, mouse plasma, and also human plasma from patients with PH-HFpEF obtained at the time of right heart catheterization. Clodronate liposomes and an anti-IL-1ß antibody were utilized to deplete macrophages and IL-1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF in mouse models. RESULTS: Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice developed PH, small vessel muscularization, and right heart dysfunction. Inflammation-related gene ontologies were overrepresented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling showed an increase in IL-1ß in mouse and human plasma. Finally, clodronate liposome treatment in mice prevented PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice, and IL-1ß depletion also attenuated PH in Nγ-nitro-L-arginine methyl ester/high-fat diet-treated mice. CONCLUSIONS: We report a novel model for the study of PH and right heart remodeling in HFpEF, and we identify myeloid cell-derived IL-1ß as an important contributor to PH in HFpEF.


Subject(s)
Heart Failure , Hypertension, Pulmonary , Animals , Female , Humans , Male , Mice , Clodronic Acid , Heart Failure/metabolism , Hypertension, Pulmonary/etiology , Interleukin-1beta , Mice, Inbred C57BL , Myeloid Cells/metabolism , Stroke Volume/physiology
2.
bioRxiv ; 2023 May 18.
Article in English | MEDLINE | ID: mdl-37292652

ABSTRACT

Background: Pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. Methods: Eight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1ß antibody treatments were utilized to deplete macrophages or IL1ß, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF. Results: Mice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1ß, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1ß was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1ß antibody treatment also attenuated PH in L-NAME/HFD treated mice. Conclusions: Our study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1ß as an important contributor to PH in HFpEF.

3.
Biomarkers ; 25(2): 131-136, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31903794

ABSTRACT

Purpose: Transpulmonary biomarkers may provide insight into pulmonary hypertension (PH) pathophysiology, but require cardiac catheterization. We investigated whether the peripheral arterial-venous ratio (PR) could substitute for the transpulmonary ratio (TPR).Materials and methods: Blood from the pulmonary artery (PA), pulmonary arterial wedge (PAW), peripheral venous, and peripheral arterial positions was analysed for ET-1, NT-pro-BNP and cAMP levels in subjects with no PH (n = 18) and PH due to left heart disease (PH-LHD), which included combined pre- and post-capillary PH (Cpc-PH; n = 7) and isolated post-capillary PH (Ipc-PH; n = 9). Bland-Altman comparisons were made between peripheral venous and PA samples and between peripheral arterial and PAW samples. TPR was defined as [PAW]/[PA].Results: For ET-1, Bland-Altman analysis indicated negative bias (-24%) in peripheral arterial compared to PAW concentration and positive bias (23%) in peripheral venous compared to PA concentration. There was <10% absolute bias for NT-pro-BNP and cAMP. For ET-1, there was no difference in PR between Cpc-PH and Ipc-PH (0.87 ± 0.4 vs. 0.94 ± 0.6, p = 0.8), whereas there was a difference in TPR (2.2 ± 1.1 vs. 1.1 ± 0.2, p < 0.05).Conclusions: In PH-LHD, peripheral samples may be inadequate surrogates for transpulmonary samples, particularly when measuring mediators with prominent pulmonary secretion or clearance, such as ET-1.


Subject(s)
Biomarkers/blood , Hypertension, Pulmonary/blood , Adult , Arteries , Blood Specimen Collection , Case-Control Studies , Cyclic AMP/blood , Endothelin-1/blood , Female , Heart Diseases/blood , Humans , Hypertension, Pulmonary/diagnosis , Hypertension, Pulmonary/physiopathology , Male , Middle Aged , Natriuretic Peptide, Brain/blood , Peptide Fragments/blood , Pulmonary Artery , Veins
4.
Am J Physiol Lung Cell Mol Physiol ; 317(4): L445-L455, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31322432

ABSTRACT

Pulmonary arterial hypertension (PAH) is a female predominant disease in which progressive vascular remodeling and vasoconstriction result in right ventricular (RV) failure and death. Most PAH patients utilize multiple therapies. In contrast, the majority of preclinical therapeutic studies are performed in male rats with a single novel drug often markedly reversing disease in the model. We sought to differentiate single drug therapy from combination therapy in female rats with severe disease. One week after left pneumonectomy, we induced PH in young female Sprague-Dawley rats with an injection of monocrotaline (45 mg/kg). Female rats were then randomized to receive combination therapy (ambrisentan plus tadalafil), ambrisentan monotherapy, tadalafil monotherapy, or vehicle. We measured RV size and function on two serial echocardiograms during the development of disease. We measured RV systolic pressure (RVSP) invasively at day 28 after monocrotaline before analyzing the vascular volume with microcomputed tomography (microCT) of the right middle lobe. RVSP was significantly lower in female rats treated with combination therapy, and combination therapy resulted in increased small vessel volume density measured by microCT compared with untreated rats. Combination-treated rats had the smallest RV end-diastolic diameter on echocardiogram as compared with the other groups. In summary, we report a female model of pulmonary hypertension that can distinguish between one and two drug therapies; this model may facilitate better preclinical drug testing for novel compounds.


Subject(s)
Antihypertensive Agents/pharmacology , Hypertension, Pulmonary/drug therapy , Hypertrophy, Right Ventricular/drug therapy , Phenylpropionates/pharmacology , Pyridazines/pharmacology , Tadalafil/pharmacology , Ventricular Dysfunction, Right/drug therapy , Animals , Disease Models, Animal , Drug Therapy, Combination/methods , Echocardiography , Female , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/diagnostic imaging , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/diagnostic imaging , Hypertrophy, Right Ventricular/physiopathology , Lung/diagnostic imaging , Lung/drug effects , Lung/physiopathology , Monocrotaline/administration & dosage , Pneumonectomy , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Rats , Rats, Sprague-Dawley , Vascular Remodeling/drug effects , Vasoconstriction/drug effects , Ventricular Dysfunction, Right/chemically induced , Ventricular Dysfunction, Right/diagnostic imaging , Ventricular Dysfunction, Right/physiopathology , X-Ray Microtomography
5.
Pulm Circ ; 9(1): 2045894018817741, 2019.
Article in English | MEDLINE | ID: mdl-30451070

ABSTRACT

Little is known about the impact of metabolic syndrome (MS) on right ventricular (RV) structure and function. We hypothesized that mice fed a Western diet (WD) would develop RV lipid accumulation and impaired RV function, which would be ameliorated with metformin. Male C57/Bl6 mice were fed a WD or standard rodent diet (SD) for eight weeks. A subset of mice underwent pulmonary artery banding (PAB). Treated mice were given 2.5 g/kg metformin mixed in food. Invasive hemodynamics, histology, Western, and quantitative polymerase chain reaction (qPCR) were performed using standard techniques. Lipid content was detected by Oil Red O staining. Mice fed a WD developed insulin resistance, RV hypertrophy, and higher RV systolic pressure compared with SD controls. Myocardial lipid accumulation was greater in the WD group and disproportionately affected the RV. These structural changes were associated with impaired RV diastolic function in WD mice. PAB-WD mice had greater RV hypertrophy, increased lipid deposition, and lower RV ejection fraction compared with PAB SD controls. Compared to untreated mice, metformin lowered HOMA-IR and prevented weight gain in mice fed a WD. Metformin reduced RV systolic pressure, prevented RV hypertrophy, and reduced RV lipid accumulation in both unstressed stressed conditions. RV diastolic function improved in WD mice treated with metformin. WD in mice leads to an elevation in pulmonary pressure, RV diastolic dysfunction, and disproportionate RV steatosis, which are exacerbated by PAB. Metformin prevents the deleterious effects of WD on RV function and myocardial steatosis in this model of the metabolic syndrome.

6.
J Pharmacol Exp Ther ; 365(3): 460-466, 2018 06.
Article in English | MEDLINE | ID: mdl-29563327

ABSTRACT

Although inhibition of phosphoinositide 3-kinase (PI3K) is an emerging strategy in cancer therapy, we and others have reported that this action can also contribute to drug-induced QT prolongation and arrhythmias by increasing cardiac late sodium current (INaL). Previous studies in mice implicate the PI3K-α isoform in arrhythmia susceptibility. Here, we have determined the effects of new anticancer drugs targeting specific PI3K isoforms on INaL and action potentials (APs) in mouse cardiomyocytes and Chinese hamster ovary cells (CHO). Chronic exposure (10-100 nM; 5-48 hours) to PI3K-α-specific subunit inhibitors BYL710 (alpelisib) and A66 and a pan-PI3K inhibitor (BKM120) increased INaL in SCN5A-transfected CHO cells and mouse cardiomyocytes. The specific inhibitors (10-100 nM for 5 hours) markedly prolonged APs and generated triggered activity in mouse cardiomyocytes (9/12) but not in controls (0/6), and BKM120 caused similar effects (3/6). The inclusion of water-soluble PIP3, a downstream effector of the PI3K signaling pathway, in the pipette solution reversed these arrhythmogenic effects. By contrast, inhibition of PI3K-ß, -γ, and -δ isoforms did not alter INaL or APs. We conclude that inhibition of cardiac PI3K-α is arrhythmogenic by increasing INaL and this effect is not seen with inhibition of other PI3K isoforms. These results highlight a mechanism underlying potential cardiotoxicity of PI3K-α inhibitors.


Subject(s)
Arrhythmias, Cardiac/chemically induced , Electrophysiological Phenomena/drug effects , Enzyme Inhibitors/pharmacology , Heart/drug effects , Heart/physiology , Phosphoinositide-3 Kinase Inhibitors , Sodium/metabolism , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/physiopathology , CHO Cells , Cricetulus , Dose-Response Relationship, Drug , Female , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism
7.
Exp Lung Res ; 44(1): 51-61, 2018 02.
Article in English | MEDLINE | ID: mdl-29381088

ABSTRACT

Purpose/Aim: Low doses (30-80 mg/kg) of monocrotaline are commonly used to create experimental models of pulmonary hypertension in rats. At these doses, monocrotaline causes pulmonary endothelial apoptosis and acute lung injury which ultimately results in pulmonary vascular disease. Higher doses of monocrotaline (300 mg/kg) are known to create severe liver injury, but previous investigations with lower doses have not reported histology in other organs to determine whether the vascular injury with monocrotaline is pulmonary-selective or generalized. MATERIALS AND METHODS: We therefore sought to determine whether monocrotaline caused extra-pulmonary injury at doses commonly used in pulmonary hypertension studies. We performed left pneumonectomy on young male and female rats before administering 50-60 mg/kg monocrotaline 7 days later. We monitored serum chemistry and urine dipsticks during the first 3 weeks while the animals developed pulmonary hypertension. After 3 weeks, we sacrificed animals and stained the lungs and highly vascular visceral organs (kidney, liver, and spleen) for elastin to evaluate the degree of vascular injury and remodeling. RESULTS: We did not observe proteinuria or significant transaminitis over the 3 weeks following monocrotaline. As previously published, monocrotaline caused severe pulmonary vascular disease with neointimal lesions and medial hypertrophy. We did not identify significant large or small arterial damage in the kidneys, liver, or spleen. Two external veterinary pathologists did not identify histopathology in the kidneys, liver, or spleen of these rats. CONCLUSIONS: We conclude that 50-60 mg/kg of monocrotaline causes a selective pulmonary vascular lesion and that male and female rats have little non-pulmonary damage over 3 weeks at these doses of monocrotaline.


Subject(s)
Monocrotaline/adverse effects , Pneumonectomy/adverse effects , Pulmonary Artery/pathology , Acute Lung Injury/chemically induced , Animals , Apoptosis/drug effects , Endothelial Cells/drug effects , Endothelial Cells/pathology , Female , Hypertension, Pulmonary/chemically induced , Lung/blood supply , Lung/pathology , Lung Diseases/chemically induced , Male , Rats
8.
Pulm Circ ; 8(1): 2045893217745019, 2018.
Article in English | MEDLINE | ID: mdl-29251543

ABSTRACT

Pulmonary hypertension complicating left heart disease (PH-LHD) is associated with increased morbidity and mortality, especially in patients who develop combined pre- and post-capillary PH (Cpc-PH). Mechanisms underlying PH-LHD are incompletely understood, particularly for individuals with preserved left ventricular ejection fraction (LVEF). We hypothesized that transpulmonary concentrations of biomarkers representing signaling pathways with known effects on the pulmonary circulation could provide insight into the molecular etiology of PH-LHD in patients with preserved LVEF. Blood samples were collected from the pulmonary artery (PA) and wedge positions of outpatients with normal LVEF referred for right heart catheterization. Hemodynamic tracings were reviewed to classify patients as "no PH" (n = 23) or "PH-LHD" (n = 22). A biomarker's transpulmonary ratio (TPR) was calculated as the quotient of wedge and PA concentrations. The TPR of endothelin 1 (ET-1) was elevated in Cpc-PH (n = 10) compared to no PH or isolated post-capillary PH (Ipc-PH, n = 12); cAMP and cGMP TPRs were not different among groups. Higher ET-1 TPR in Cpc-PH was due to increased wedge ET-1 concentration. Pulmonary vascular resistance (PVR) strongly correlated with wedge ET-1 exclusively in Cpc-PH patients. In patients with normal LVEF and Cpc-PH, ET-1 TPR is higher, due to elevated wedge ET-1, compared to those without PH or with Ipc-PH. Strong correlation between PVR and wedge ET-1, observed only in the Cpc-PH group, may suggest increased pulmonary vascular responsiveness to ET-1 in these patients. These findings implicate elevated pulmonary ET-1 as a marker of, and a potential contributor to, development of Cpc-PH in this population.

9.
Can J Physiol Pharmacol ; 88(8): 830-9, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20725141

ABSTRACT

Endothelin 1 (ET-1) is an endogenous peptide that promotes vasoconstriction, endothelial and smooth muscle cell (SMC) proliferation, and fibrosis. ET-1 receptor antagonists are an important treatment strategy for pulmonary arterial hypertension, but less effective in systemic vascular disease. This observation suggests a special role for ET-1 in the pulmonary circulation. We hypothesized that ET-1 contributes to the pathogenesis of pulmonary arterial hypertension, in part, by promoting pulmonary vascular SMC migration. ET-1 treatment promoted migration in 3 distinct types of cultured pulmonary SMC. Pulmonary SMC migration was blocked by an ETA receptor selective agonist and a combined ETA-ETB antagonist, but not by a selective ETB antagonist. In contrast to the effect on pulmonary SMCs, ET-1 had no effect on migration of aortic SMCs. Flow cytometry showed that the ETA receptor was expressed at comparable levels on pulmonary and aortic SMCs, excluding receptor density as an explanation for the divergent effect. ET-1-induced pulmonary SMC migration was blocked by the structurally distinct MEK inhibitors PD98059 and U0126, consistent with a role for ERK1/2 MAP kinase. By Western blot in cultured cells and immunohistochemistry in ex vivo vessels, ET-1 stimulated phosphorylation of ERK1/2 as efficaciously as platelet-derived growth factor in pulmonary, but not aortic, SMCs. In conclusion, ET-1 induces SMC migration, with the ETA receptor tightly coupled to ERK1/2 phosphorylation only in the pulmonary circulation. This finding may help explain the striking difference in the efficacy of endothelin receptor blockers for pulmonary hypertension as compared to that for systemic cardiovascular disease.


Subject(s)
Aorta/cytology , Cell Movement/physiology , Endothelin-1/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/cytology , Animals , Antihypertensive Agents/pharmacology , Aorta/drug effects , Aorta/metabolism , Becaplermin , Cell Line , Cell Movement/drug effects , Cells, Cultured , Endothelin A Receptor Antagonists , Endothelin B Receptor Antagonists , Humans , Male , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Oligopeptides/pharmacology , Peptides, Cyclic/pharmacology , Phosphorylation/drug effects , Platelet-Derived Growth Factor/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-sis , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Endothelin A/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Tunica Media/drug effects , Tunica Media/metabolism
10.
JACC Cardiovasc Imaging ; 2(10): 1198-208, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19833310

ABSTRACT

OBJECTIVES: The purpose of this study was to compare a selective A(2A) adenosine receptor agonist (regadenoson) with adenosine in clinically relevant canine models with regard to effects on hemodynamics and thallium-201 ((201)Tl) and technetium-99m ((99m)Tc)-sestaMIBI biodistribution and kinetics. BACKGROUND: The clinical application of vasodilator stress for perfusion imaging requires consideration of the effects of these vasodilating agents on systemic hemodynamics, coronary flow, and radiotracer uptake and clearance kinetics. METHODS: Sequential imaging and arterial blood sampling was performed on control, anesthetized closed-chest canines (n = 7) to evaluate radiotracer biodistribution and kinetics after either a bolus administration of regadenoson (2.5 microg/kg) or 4.5-min infusion of adenosine (280 microg/kg). The effects of regadenoson on coronary flow and myocardial radiotracer uptake were then evaluated in an open-chest canine model of a critical stenosis (n = 7). Results from ex vivo single-photon emission computed tomography were compared with tissue well-counting. RESULTS: The use of regadenoson compared favorably with adenosine in regard to the duration and magnitude of the hemodynamic effects and the effect on (201)Tl and (99m)Tc-sestaMIBI biodistribution and kinetics. The arterial blood clearance half-time was significantly faster for (99m)Tc-sestaMIBI (regadenoson: 1.4 +/- 0.03 min; adenosine: 1.5 +/- 0.08 min) than for (201)Tl (regadenoson: 2.5 +/- 0.16 min, p < 0.01; adenosine: 2.7 +/- 0.04 min, p < 0.01) for both vasodilator stressors. The relative microsphere flow deficit (0.34 +/- 0.02%) during regadenoson stress was significantly greater than the relative perfusion defect with (99m)Tc-sestaMIBI (0.69 +/- 0.03%, p < 0.001) or (201)Tl (0.53 +/- 0.02%, p < 0.001), although (201)Tl tracked the flow deficit within the ischemic region better than (99m)Tc-sestaMIBI. The perfusion defect score was larger with (201)Tl (22 +/- 2.8% left ventricular) than with (99m)Tc-sestaMIBI (17 +/- 1.7% left ventricular, p < 0.05) on ex vivo single-photon emission computed tomography images. CONCLUSIONS: The bolus administration of regadenoson produced a hyperemic response comparable to a standard infusion of adenosine. The biodistribution and clearance of both (201)Tl and (99m)Tc-sestaMIBI during regadenoson were similar to adenosine vasodilation. Ex vivo perfusion images under the most ideal conditions permitted detection of a critical stenosis, although (201)Tl offered significant advantages over (99m)Tc-sestaMIBI for perfusion imaging during regadenoson vasodilator stress.


Subject(s)
Adenosine A2 Receptor Agonists , Adenosine/pharmacology , Coronary Circulation/drug effects , Coronary Stenosis/diagnostic imaging , Myocardial Perfusion Imaging/methods , Purines/pharmacology , Pyrazoles/pharmacology , Radiopharmaceuticals/pharmacokinetics , Technetium Tc 99m Sestamibi/pharmacokinetics , Thallium Radioisotopes/pharmacokinetics , Tomography, Emission-Computed, Single-Photon , Vasodilator Agents/pharmacology , Acute Disease , Adenosine/administration & dosage , Animals , Blood Flow Velocity/drug effects , Chronic Disease , Coronary Stenosis/metabolism , Coronary Stenosis/physiopathology , Disease Models, Animal , Dogs , Hyperemia/diagnostic imaging , Hyperemia/physiopathology , Infusions, Intravenous , Injections, Intravenous , Metabolic Clearance Rate , Myocardium/metabolism , Myocardium/pathology , Predictive Value of Tests , Purines/administration & dosage , Pyrazoles/administration & dosage , Radiopharmaceuticals/blood , Receptor, Adenosine A2A/metabolism , Technetium Tc 99m Sestamibi/blood , Thallium Radioisotopes/blood , Tissue Distribution , Vasodilator Agents/administration & dosage
11.
Am J Physiol Lung Cell Mol Physiol ; 297(4): L706-14, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19648282

ABSTRACT

Pulmonary arterial hypertension (PAH) is a progressive disease of excess vasoconstriction and vascular cell proliferation that results in increased pulmonary vascular resistance and right heart failure. We have previously shown (66) that tissue factor expression is increased in the abnormal vessels of patients and rats with PAH. We hypothesized that tissue factor and its downstream mediator, thrombin, would promote migration of endothelial cells (EC) and the vascular pathology of PAH. Immunostaining revealed EC and a fibronectin-enriched matrix within the "plexiform-like" lesions in a rat model of severe PAH. In a modified Boyden assay, protease-activated receptor 1 (PAR1; thrombin receptor) stimulation by agonist peptide or thrombin induced pulmonary microvascular EC (PMVEC) migration when the cells were interacting with fibronectin, but not with other extracellular matrix proteins. Thrombin/fibronectin-induced migration was confirmed in wound healing and angiogenesis assays and was abrogated by the PAR1 antagonist SCH79797 and soluble RGD peptide. This fibronectin dependence was unique to PAR1 activation; other EC agonists evaluated did not induce migration on any matrix, and 10% FBS stimulated similar levels of migration on all matrix proteins tested. Thrombin/fibronectin stimulated autophosphorylation of calcium/calmodulin dependent protein kinase II (CaMKII) in PMVEC, and inhibitors of CaMKII blocked thrombin-induced migration on fibronectin, but had no effect on migration induced by 10% FBS. In contrast, EC isolated from the proximal pulmonary artery migrated in response to most agonists independent of the matrix substrate. Our findings illustrate EC heterogeneity in a single tissue and indicate a novel role for CaMKII in mediating EC migration. Because PMVEC have been shown to have impressive proliferative potential, thrombin/fibronectin-stimulated migration of these cells to a site of injured endothelium is a potential mechanism by which thrombin contributes to the development of vascular lesions in PAH.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Movement/drug effects , Endothelium, Vascular/drug effects , Fibronectins/pharmacology , Hemostatics/pharmacology , Pulmonary Artery/drug effects , Thrombin/pharmacology , Animals , Blotting, Western , Cells, Cultured , Endothelium, Vascular/cytology , Oligopeptides/pharmacology , Pulmonary Artery/cytology , Rats , Receptor, PAR-1/metabolism , Wound Healing/drug effects
12.
J Appl Physiol (1985) ; 104(5): 1504-12, 2008 May.
Article in English | MEDLINE | ID: mdl-18356482

ABSTRACT

The alphavbeta3-integrin is expressed in angiogenic vessels in response to hypoxia and represents a potential novel target for imaging myocardial angiogenesis. This study evaluated the feasibility of noninvasively tracking hypoxia-induced alphavbeta3-integrin activation within the myocardium as a marker of angiogenesis early after myocardial infarction. Acute myocardial infarction was produced by coronary artery occlusion in rodent and canine studies. A novel (111)In-labeled radiotracer targeted at the alphavbeta3-integrin ((111)In-RP748) was used to localize regions of hypoxia-induced angiogenesis early after infarction. In rodent studies, the specificity of (111)In-RP748 for alphavbeta3-integrin was confirmed with a negative control compound ((111)In-RP790), and regional uptake of these compounds correlated with (201)Tl perfusion and a (99m)Tc-labeled nitroimidazole (BRU59-21), which was used as a quantitative marker of myocardial hypoxia. The ex vivo analysis demonstrated that only (111)In-RP748 was selectively retained in infarcted regions with reduced (201)Tl perfusion and correlated with uptake of BRU59-21. In canine studies, myocardial uptake of (111)In-RP748 was assessed using in vivo single-photon-emission computed tomography (SPECT), ex vivo planar imaging, and gamma well counting of myocardial tissue and correlated with (99m)Tc-labeled 2-methoxy-2-methyl-propyl-isonitrile ((99m)Tc-sestamibi) perfusion. Dual-radiotracer in vivo SPECT imaging of (111)In-RP748 and (99m)Tc-sestamibi provided visualization of (111)In-RP748 uptake within the infarct region, which was confirmed by ex vivo planar imaging of excised myocardial slices. Myocardial (111)In-RP748 retention was associated with histological evidence of alphavbeta3-integrin expression/activation in the infarct region. (111)In-RP748 imaging provides a novel noninvasive approach for evaluation of hypoxia-induced alphavbeta3-integrin activation in myocardium early after infarction and may prove useful for directing and evaluating angiogenic therapies in patients with ischemic heart disease.


Subject(s)
Hypoxia/metabolism , Integrins/metabolism , Myocardial Infarction/metabolism , Myocardium/metabolism , Actins/biosynthesis , Actins/genetics , Animals , Biomarkers , Biotransformation/physiology , Dogs , Heterocyclic Compounds, 1-Ring , Imidazoles , Immunohistochemistry , Integrin alphaVbeta3/biosynthesis , Integrin alphaVbeta3/genetics , Male , Myocardial Infarction/pathology , Myocardial Ischemia/pathology , Myocardium/pathology , Neovascularization, Physiologic/physiology , Organometallic Compounds , Organotechnetium Compounds , Radiopharmaceuticals , Rats , Rats, Sprague-Dawley , Technetium Tc 99m Sestamibi , Tomography, Emission-Computed, Single-Photon
13.
Am J Physiol Lung Cell Mol Physiol ; 293(3): L583-90, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17586694

ABSTRACT

Severe pulmonary arterial hypertension (PAH) occurs in idiopathic form and in association with diverse diseases. The pathological hallmarks are distal smooth muscle hypertrophy, obliteration of small pulmonary arteriole lumens, and disorganized cellular proliferation in plexiform lesions. In situ thrombosis is also observed. A detailed understanding of the disease progression has been hampered by the absence of an animal model bearing all the pathological features of human disease. To create a model with these characteristics, we gave young (200-g) rats monocrotaline 1 wk following left pneumonectomy; controls with vehicle treatment or sham operation were also studied. In experimental rats, pulmonary arteries had distal smooth muscle hypertrophy and proliferative perivascular lesions. The lesions had a plexiform appearance, occurred early in disease development, and were composed of cells expressing endothelial antigens. Three-dimensional microangiography revealed severe vascular pruning and disorganized vascular networks. We found that expression of tissue factor (TF), the membrane glycoprotein that initiates coagulation, facilitates angiogenesis, and mediates arterial injury in the systemic circulation, was increased in the pulmonary arterioles and plexiform-like lesions of the rats. TF was also heavily expressed in the vessels and plexiform lesions of humans with pulmonary arterial hypertension. We conclude that plexiform-like lesions can be reproduced in rats, and this model will facilitate experiments to address controversies about the role of these lesions in PAH. Increased TF expression may contribute to the prothrombotic diathesis and vascular cell proliferation typical of human disease.


Subject(s)
Disease Models, Animal , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Thromboplastin/metabolism , Angiography , Animals , Cell Proliferation/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Humans , Hypertension, Pulmonary/chemically induced , Hypertrophy, Right Ventricular/chemically induced , Hypertrophy, Right Ventricular/pathology , Male , Monocrotaline/administration & dosage , Monocrotaline/pharmacology , Pneumonectomy , Pulmonary Artery/diagnostic imaging , Pulmonary Artery/drug effects , Rats , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor Receptor-2/metabolism , von Willebrand Factor/metabolism
14.
Circ Res ; 98(6): 811-7, 2006 Mar 31.
Article in English | MEDLINE | ID: mdl-16527992

ABSTRACT

Reactive oxygen species (ROS) contribute to the pathogenesis of atherosclerosis in part by promoting vascular smooth muscle cell (VSMC) growth. Previously we demonstrated that cyclophilin A (CyPA) is a secreted oxidative stress-induced factor (SOXF) that promotes inflammation, VSMC growth, and endothelial cell apoptosis. However, the mechanisms that regulate CyPA secretion are unknown. In this study, we hypothesized that ROS-induced CyPA secretion from VSMC requires a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane. Conditioned medium and plasma membrane sheets were prepared by exposing VSMC to 1 micromol/L LY83583, which generates intracellular superoxide. A vesicular transport mechanism was confirmed by colocalization at the plasma membrane with vesicle-associated membrane protein (VAMP). CyPA transport to the plasma membrane and secretion were significantly increased by LY83583. Reduction of VAMP-2 expression by small interfering RNA inhibited LY83583-induced CyPA secretion. Pretreatment with 3 micromol/L cytochalasin D, an actin depolymerizing agent, abrogated CyPA secretion. Infection with dominant-negative RhoA and Cdc42 adenovirus inhibited CyPA secretion by 72% and 63%, respectively, whereas dominant-negative Rac1 had a small effect (11%). Pretreatment with the Rho kinase inhibitor Y27632 (3 to 30 micromol/L) and myosin II inhibitor blebbistatin (1 to 10 micromol/L) inhibited CyPA secretion in a dose-dependent manner. Simvastatin (3 to 30 micromol/L) also dose-dependently inhibited LY83583-induced CyPA secretion likely via decreased isoprenylation of small GTPases. Our findings define a novel VSMC vesicular secretory pathway for CyPA that involves actin remodeling and myosin II activation via RhoA-, Cdc42-, and Rho kinase-dependent signaling events.


Subject(s)
Cyclophilin A/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Actins/metabolism , Aminoquinolines/pharmacology , Animals , Cell Membrane/metabolism , Intracellular Signaling Peptides and Proteins , Male , Muscle, Smooth, Vascular/cytology , Myosin Type II/physiology , Oxidative Stress , Protein Serine-Threonine Kinases/physiology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/toxicity , Simvastatin/pharmacology , Vesicle-Associated Membrane Protein 2/physiology , rho GTP-Binding Proteins/physiology , rho-Associated Kinases
15.
J Clin Invest ; 113(12): 1684-91, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15199403

ABSTRACT

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.


Subject(s)
Coronary Vessels/metabolism , Diagnostic Imaging , Integrin alphaVbeta3/metabolism , Myocardial Infarction , Myocardium/metabolism , Neovascularization, Physiologic , Animals , Cells, Cultured , Coronary Vessels/anatomy & histology , Dogs , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Hemodynamics , Indium Radioisotopes/chemistry , Indium Radioisotopes/metabolism , Male , Molecular Structure , Quinolones/chemistry , Quinolones/metabolism , Radiopharmaceuticals/metabolism , Rats , Rats, Sprague-Dawley , Technetium Tc 99m Sestamibi/metabolism , Tomography, Emission-Computed, Single-Photon
16.
Nat Med ; 8(12): 1427-32, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12415262

ABSTRACT

The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa(164) cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect.


Subject(s)
Angiogenesis Inducing Agents , Gene Expression Regulation , Neovascularization, Physiologic , Protein Engineering/methods , Transcription Factors/physiology , Vascular Endothelial Growth Factor A , Zinc Fingers/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Drug Design , Genetic Therapy , Mice , Models, Animal , Molecular Sequence Data , Recombinant Proteins , Transcription Factors/genetics
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