Abstract-Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization. We hypothesized that Ang-1 gene transfer to the pulmonary microcirculation would improve pulmonary hemodynamics and vascular remodeling in experimental pulmonary hypertension. Rat pulmonary artery smooth muscle cells were transfected with Ang-1 cDNA or null (pFLAG-CMV-1) vector. Syngeneic Fisher 344 rats were treated with monocrotaline (MCT) (75 mg/kg IP) with or without delivery of 5ϫ10 5 Ang-1-transfected cells into the right jugular vein. After 28 days, plasmid-derived Ang-1 mRNA was consistently and robustly detected by reverse transcriptase-polymerase chain reaction in lungs from all animals receiving Ang-1 gene therapy. Tie-2 receptor expression was markedly downregulated in rats treated with MCT, and this was partially restored by gene therapy with Ang-1. Animals receiving MCT exhibited 77% mortality by 28 days. In contrast, in pAng-1-treated animals, the 28-day mortality was only 14% (PϽ0.0001). In addition, right ventricular systolic pressure was reduced from 52Ϯ1.3 mm Hg in the MCT-treated group to 38Ϯ1.3 mm Hg by Ang-1 gene transfer (PϽ0.01), whereas the measurement of right to left ventricular plus septal weight ratio was also reduced from 0.41Ϯ0.03 to 0.31Ϯ0.01 (PϽ0.05). Moreover, MCT resulted in increased apoptosis, mainly in the microvasculature, and reduced endothelial NO synthase mRNA expression, both of which were prevented by Ang-1 gene transfer. Thus, cell-based gene transfer with Ang-1 improved survival and pulmonary hemodynamics in experimental pulmonary hypertension by a mechanism involving the inhibition of apoptosis and protection of the pulmonary microvasculature.

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.

Abstract-Endothelium-derived NO plays a critical role in the regulation of cardiovascular function and structure, as well as acting as a downstream mediator of the angiogenic response to numerous vascular growth factors. Although endothelial NO synthase (eNOS)-deficient mice are viable, minor congenital cardiac abnormalities have been reported and homozygous offspring exhibit high neonatal mortality out of proportion to the severity of these defects. The aim of the present report was to determine whether abnormalities of the pulmonary vascular development could contribute to high neonatal loss in eNOS-deficient animals. We now report that eNOS-deficient mice display major defects in lung morphogenesis, resulting in respiratory distress and death within the first hours of life in the majority of animals. Histological and molecular examination of preterm and newborn mutant lungs demonstrated marked thickening of saccular septae, with evidence of reduced surfactant material. Lungs of eNOS-deficient mice also exhibited a striking paucity of distal arteriolar branches and extensive regions of capillary hypoperfusion, together with misalignment of pulmonary veins, which represent the characteristic features of alveolar capillary dysplasia. We conclude that eNOS plays a previously unrecognized role in lung development, which may have relevance for clinical syndromes of neonatal respiratory distress. Key Words: nitric oxide Ⅲ angiogenesis Ⅲ surfactant Ⅲ respiratory distress syndrome Ⅲ alveolar capillary dysplasia N O is a multifaceted vasodilator that has also been shown to be an important regulator of vascular growth and remodeling. 1 NO has been implicated as a critical downstream mediator in the biological response to a variety of angiogenic growth factors, 2-5 mediating endothelial cell (EC) proliferation, 6 migration, 7 and vascular tube formation. 3 Adult eNOS-deficient mice (eNOS Ϫ/Ϫ ) exhibit marked defects in postnatal angiogenesis, 8 moderate systemic hypertension, 9 and mild elevation in pulmonary vascular resistance with an exaggerated pulmonary vasoconstrictor response to hypoxia. 10 Although eNOS Ϫ/Ϫ mice are viable, litter sizes are characteristically small, and these offspring exhibit minor abnormalities such as bicuspid aortic valve 11 and atrial septal defects. 12 eNOS is strongly expressed in rodent 13 and ovine 14 fetal lungs at mid to late gestation, in both vascular endothelial as well as airway epithelial cells, 15 consistent with a possible role in the regulation of pulmonary vasculature and airway development. However, to date there has been no direct evidence implicating NO in fetal lung development, although recently eNOS has been shown to be involved in compensatory hyperplasia of postnatal lung 16 and in preventing loss of alveolarization during neonatal exposure to hypoxia. 17 The paradox between the important role of NO in postnatal angiogenesis, yet the apparently normal embryonic vascular development in eNOS Ϫ/Ϫ animals could be explained by a high degree of redundancy, which often is as...