Hilde Gillijns

Increased Cardiac Expression of Tissue Inhibitor of Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-2 Is Related to Cardiac Fibrosis and Dysfunction in the Chronic Pressure-Overloaded Human Heart

Background—Alterations in the balance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) are involved in left ventricular (LV) remodeling. Whether their expression is related to interstitial fibrosis or LV dysfunction in patients with chronic pressure overload–induced LV hypertrophy, however, is unknown. Methods and Results—Therefore, cardiac biopsies were taken in 36 patients with isolated aortic stenosis (AS) and in 29 control patients without LV hypertrophy. Microarray analysis revealed significantly increased mRNA expression of collagen types I, III, and IV and transcripts involved in collagen synthesis, including procollagen endopeptidase and lysine and proline hydroxylases, in AS compared with control patients. Collagen deposition was greater in AS than in control patients and was most pronounced in AS patients with severe diastolic dysfunction. Cardiac mRNA expression of TIMP-1 and TIMP-2 was significantly increased in AS compared with control patients (mRNA transcript levels normalized to GAPDH: TIMP-1, 0.67±0.1 in AS versus 0.37±0.08 in control patients; TIMP-2, 9.5±2.6 in AS versus 1.6±0.4 in control patients; P<0.05 for both) but did not differ significantly for MMP-1, -2, or -9. Cardiac TIMP-1 and -2 transcripts were significantly related to the degree of interstitial fibrosis and proportional to diastolic dysfunction in AS patients. Conclusions—Cardiac expression of TIMP-1 and TIMP-2 is significantly increased in chronic pressure-overloaded human hearts compared with controls and is related to the degree of interstitial fibrosis.

Cardiomyocyte-Specific Overexpression of Nitric Oxide Synthase 3 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction

Nitric oxide (NO) is an important modulator of cardiac performance and left ventricular (LV) remodeling after myocardial infarction (MI). We tested the effect of cardiomyocyte-restricted overexpression of one NO synthase isoform, NOS3, on LV remodeling after MI in mice. LV structure and function before and after permanent LAD coronary artery ligation were compared in transgenic mice with cardiomyocyte-restricted NOS3 overexpression (NOS3-TG) and their wild-type littermates (WT). Before MI, systemic hemodynamic measurements, echocardiographic assessment of LV fractional shortening (FS), heart weight, and myocyte width (as assessed histologically) did not differ in NOS3-TG and WT mice. The inotropic response to graded doses of isoproterenol was significantly reduced in NOS3-TG mice. One week after LAD ligation, the infarcted fraction of the LV did not differ in WT and NOS3-TG mice (34 ± 4% versus 36 ± 12%, respectively). Four weeks after MI, however, end-systolic LVID was greater, and fractional shortening and maximum and minimum rates of LV pressure development were less in WT than in NOS3-TG mice. LV weight/body weight ratio was greater in WT than in NOS3-TG mice (5.3 ± 0.2 versus 4.6 ± 0.5 mg/g; P < 0.01). Myocyte width in noninfarcted myocardium was greater in WT than in NOS3-TG mice (18.8 ± 2.0 versus 16.6 ± 1.6 μm; P < 0.05), whereas fibrosis in noninfarcted myocardium was similar in both genotypes. Cardiomyocyte-restricted overexpression of NOS3 limits LV dysfunction and remodeling after MI, in part by decreasing myocyte hypertrophy in noninfarcted myocardium.

Local Adenovirus-Mediated Transfer of Human Endothelial Nitric Oxide Synthase Reduces Luminal Narrowing After Coronary Angioplasty in Pigs

BACKGROUND Nitric oxide, synthesized from L-arginine by nitric oxide synthase (NOS), is a vasodilator and inhibits vascular smooth muscle cell (SMC) proliferation and migration. The effects of local NOS gene transfer on restenosis after experimental balloon angioplasty were investigated. METHODS AND RESULTS Left anterior descending coronary artery angioplasty was performed in 25 pigs. Animals received an intramural injection of adenovirus (1.5 x 10(9) pfu) carrying either the NOS cDNA (AdCMVceNOS) or no cDNA (AdRR5) via the Infiltrator. Local gene transfer efficiency and bioactivity of recombinant protein were assessed after 4 days. Indices of restenosis were evaluated by computerized planimetry on coronary artery sections prepared 28 days after angioplasty. Adenoviral vectors permitted efficient gene delivery to medial SMCs and adventitial cells of coronary arteries. Vascular cGMP levels were depressed after angioplasty from 1.30+/-0.42 to 0.33+/-0.20 pmol/mg protein (P<0.05) but were restored after constitutive endothelial (ce) NOS gene transfer to 1.82+/-0.98 pmol/mg (P<0.05 versus injured group and P=NS versus control). The ratio of the neointimal area to the internal elastic lamina fracture length, maximal neointimal thickness, and percent stenosis were all reduced in AdCMVceNOS- versus AdRR5-transduced pigs (0.59+/-0.14 versus 0.80+/-0.19 mm, P=0.02; 0.75+/-0.21 versus 1.04+/-0.25 mm, P=0.019; and 53+/-15% versus 75+/-11%, P=0.006, respectively). Lumen area was significantly larger (0.70+/-0.35 mm2 in AdCMVceNOS versus 0.32+/-0.18 mm2 in AdRR5, P=0.007). CONCLUSIONS Percutaneous adenovirus-mediated NOS gene transfer resulted in efficient local overexpression of functional NOS after angioplasty in coronary arteries. Restored NO production in injured coronary arteries significantly reduced luminal narrowing, most likely through a combined effect on neointima formation and on vessel remodeling after angioplasty.

Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice

Background— Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). Methods and Results— Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57±5 versus 39±4 and 65±6 versus 48±4 &mgr;L, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. Conclusions— Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.

Cytochrome P450 Epoxygenase Gene Function in Hypoxic Pulmonary Vasoconstriction and Pulmonary Vascular Remodeling

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase–derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (−46% and −55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

Aerosol Gene Transfer With Inducible Nitric Oxide Synthase Reduces Hypoxic Pulmonary Hypertension and Pulmonary Vascular Remodeling in Rats

BackgroundNitric oxide (NO) is a potent vasodilator with an important role in the regulation of pulmonary vascular tone. The effects of NO synthase (NOS) gene transfer on pulmonary vascular remodeling associated with hypoxic pulmonary hypertension are unknown. Methods and ResultsWe aerosolized 3×109 pfu of an adenoviral vector containing inducible NOS gene (AdNOS2), constitutive NOS3 gene (AdNOS3), or no transgene (AdRR5) into rat lungs. Exhaled NO levels, monitored with chemiluminescence, were higher in AdNOS2-infected rats than in AdNOS3- and AdRR5-infected rats (at 3 days, 33±6 ppb, n=9, versus 17±4, n=9, and 6±2 ppb, n=3, P <0.05 for both). Exposure to Fio2 0.10 for 7 days increased pulmonary artery pressure from 19±4 mm Hg (baseline) to 27±1 and 26±2 mm Hg in AdNOS3- and AdRR5-infected rats, respectively, but only to 21±1 mm Hg in AdNOS2-infected animals (P <0.05). After 7 days of hypoxia, total pulmonary resistance in AdRR5- and AdNOS3-infected rats was significantly higher than in AdNOS2-infected animals (0.41±0.05 and 0.39±0.07 versus 0.35±0.03 mm Hg · mL−1 · min−1, respectively, P <0.05). Right ventricular hypertrophy was reduced in AdNOS2-infected rats [right ventricular/(left ventricular+septal) weight, 0.19±0.10 versus 0.28±0.10 and 0.32±0.10 in AdRR5- and AdNOS3-infected rats, respectively, P <0.05]. The percentage of muscularized precapillary pulmonary resistance vessels was also significantly decreased (18±4% versus 25±8% and 30±5% in AdRR5- and AdNOS3-infected rats, P <0.05). ConclusionsAerosol NOS2 gene transfer increases pulmonary NO production and significantly reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling. Aerosol NOS2 gene transfer may be a promising strategy to target pulmonary vascular disorders.

Sildenafil improves exercise hemodynamics in Fontan patients.

Background—Patients with Fontan circulation have reduced exercise capacity. The absence of a presystemic pump may limit flow through the pulmonary circulation, restricting ventricular filling and cardiac output. We evaluated exercise hemodynamics and the effect of sildenafil on exercise hemodynamics in Fontan patients. Methods and Results—Ten Fontan patients (6 men, 20±4 years) underwent cardiac magnetic resonance imaging at rest and during supine bicycle exercise before and after sildenafil. Systemic ventricular volumes were obtained at rest and during low- (34±15 W), moderate- (69±29 W), and high-intensity (97±36 W) exercise using an ungated, free-breathing cardiac magnetic resonance sequence and analyzed correcting for cardiac phase and respiratory translation. Radial and pulmonary artery pressures and cGMP were measured. Before sildenafil, cardiac index increased throughout exercise (4.0±0.9, 5.9±1.1, 7.0±1.6, 7.4±1.7 L/(min·m2); P<0.0001) with 106±49% increase in heart rate. Stroke volume index (P=0.015) and end-diastolic volume index (P=0.001) decreased during exercise. End-systolic volume index remained unchanged (P=0.8). Total pulmonary resistance index (P=0.005) increased, whereas systemic vascular resistance index decreased during exercise (P<0.0001). Sildenafil increased cardiac index (P<0.0001) and stroke volume index (P=0.003), especially at high-intensity exercise (interaction P=0.004 and P=0.003, respectively). Systemic vascular resistance index was reduced (P<0.0001–interaction P=0.1), whereas total pulmonary resistance index was reduced at rest and reduced further during exercise (P=0.008–interaction P=0.029). cGMP remained unchanged before sildenafil (P=0.9), whereas it increased significantly after sildenafil (P=0.019). Conclusions—In Fontan patients, sildenafil improved cardiac index during exercise with a decrease in total pulmonary resistance index and an increase in stroke volume index. This implies that pulmonary vasculature represents a physiological limitation, which can be attenuated by sildenafil, the clinical significance of which warrants further study.

Soluble Guanylate Cyclase-α1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia

Background— Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of &agr;- and &bgr;-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional &agr;1-subunit (sGC&agr;1−/−). Methods and Results— sGC&agr;1−/− mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22±2 to 34±2 mm Hg) and sGC&agr;1−/− (23±2 to 34±1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22±2 to 24±2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGC&agr;1−/− mice (22±1 to 33±1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGC&agr;1−/− mice as measured by telemetry (−37±2 versus −42±4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGC&agr;1−/− than in WT mice (P<0.01). Increased remodeling in sGC&agr;1−/− mice was associated with an increased frequency of 5′-bromo-deoxyuridine–positive vessels after 1 and 3 weeks (P<0.01 versus WT). Conclusions— Deficiency of sGC&agr;1 does not alter hypoxic pulmonary vasoconstriction. sGC&agr;1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Overexpression of a Constitutively Active Protein Kinase G Mutant Reduces Neointima Formation and In-Stent Restenosis

Background—Neointima formation after arterial injury is associated with reduced vascular cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), a major cGMP effector in vascular smooth muscle. We tested the effect of PKG overexpression on the neointimal response to vascular injury. Methods and Results—Infection of cultured rat aortic smooth muscle cells (RASMCs) with an adenoviral vector specifying a cGMP-independent, constitutively active PKG mutant (AdPKGcat) reduced serum-induced migration by 33% and increased serum-deprivation–induced apoptosis 2-fold (P <0.05 for both). Infection with wild-type PKG (AdPKG), in the absence of cGMP, did not affect migration or apoptosis. Two weeks after balloon-injured rat carotid arteries were infected with 1× 1010 pfu AdPKGcat (n=12), AdPKG (n=8), or a control adenovirus (n=8), intima-to-media ratio was less in AdPKGcat-infected arteries than in AdPKG- or control adenovirus–infected vessels (0.26±0.06 versus 0.61±0.12 and 0.70±0.12, respectively, P <0.05 for both). Two weeks after intramural administration of 1.75×1010 pfu AdPKGcat (n=8) or a control adenovirus (n=8) into porcine coronary arteries with in-stent restenosis, luminal diameter was greater in AdPKGcat-infected arteries than in control adenovirus-infected vessels (2.32±0.16 versus 1.81±0.13 mm, P =0.028), associated with reduced neointimal area (3.30±0.24 versus 4.15±0.13 mm2, P =0.008), neointima-to-vessel area ratio (0.42±0.05 versus 0.58±0.04, P <0.05), and percent stenosis (45±6% versus 70±4%, P <0.05). Conclusions—Expression of a constitutively active PKG reduces neointima formation after balloon injury in rats and reduces coronary in-stent restenosis in pigs. PKGcat gene transfer may be a promising strategy for vasculoproliferative disorders.

Percutaneous adenoviral gene transfer into porcine coronary arteries : Is catheter-based gene delivery adapted to coronary circulation?

Recombinant adenoviral (Ad) vectors represent an efficient gene transfer system for targeting the cardiovascular system. Phenotypic modulation of coronary vascular cells in vivo is, however, critically dependent on the efficacy of local delivery devices. Four local drug delivery catheters were tested for intracoronary gene transfer efficiency: the Infiltrator (INF, n = 10), the Crescendo (CRE, n = 10), the Infusasleeve (SLE, n = 8), and the Remedy balloon (channel balloon [CHA], n = 8). After balloon injury of the LAD, Ad vector containing the firefly luciferase cDNA (AdCMVluc, 1.5 x 10(10) plaque-forming units) was administered at the site of injury. On day 4, tissue samples from different regions in the heart and from the liver were assayed for luciferase activity to evaluate local and systemic gene transfer. INF, CRE, and SLE catheters showed higher transduction levels of the target LAD segment than did the CHA catheter (median luciferase activity = 4.2 x 10(6), 11 x 10(6), and 1.3 x 10(6) light units [LU]/vessel versus 0.09 x 10(6) LU/vessel, respectively, p < 0.05). Luciferase activity was occasionally observed in nontarget tissues (right and left ventricular free wall, distal LAD, and liver) and was not significantly different between groups. The viral circulatory half-life was similar for the four groups (<1 min). Gene transfer efficiency was positively correlated with the degree of injury for the intralumenal catheters (CRE, SLE, and CHA) but was independent of the vessel wall injury for the intramural INF. Local drug delivery catheters enable efficient vascular gene transfer in balloon-injured coronary arteries, a prerequisite for further development of intracoronary gene therapy for restenosis.