Pulmonary Vascular Growth Research Articles

Evaluation of pulmonary GLUT1 and VEGF mRNA levels in relation to lung weight in medicolegal autopsy cases

The present study focuses attention upon the relationship among postmortem mRNA levels of pulmonary glucose transporter (GLUT1) and vascular endothelial growth factor (VEGF) to lung weight to investigate pulmonary pathophysiology in the death process. Autopsy cases ( n = 173, within 48 h postmortem) of blunt injury, including head injury (brain contusions and acute subdural hemotoma) and non-head injury, sharp instrument injury, mechanical asphyxiation, drowning, acute myocardial infarction/ischemia (AMI) and idiopathic cerebral hemorrhage (ICH) were examined. GLUT1 and VEGF mRNAs were quantified by TaqMan real-time RT-PCR for the upper lobes of the bilateral lungs. Combined lung weight was normalized against height for statistical analyses. GLUT1 mRNA showed a higher level for ICH. GLUT1 and VEGF mRNA levels were higher for brain contusions than for acute subdural hematoma, which showed a significantly lower VEGF mRNA level. Lung weight showed a larger value for saltwater drowning and ICH, and was larger for acute subdural hematoma than for brain contusions. GLUT1 mRNA level was correlated with lung weight in cases of ICH and brain contusions (survival time <24 h), and VEGF mRNA showed a similar tendency. Such findings were not detected for other groups. These findings indicate parallel increases in hypoxia-induced responses and lung weight in ICH and brain contusions, suggesting different pulmonary hemodynamics with milder alveolar damage compared with other groups, including AMI and acute subdural hematoma. Different mechanisms might be involved in non-cardiogenic or neurogenic pulmonary congestion and edema for ICH/brain contusions and subdural hematoma.

Atrasentan Treatment of Pulmonary Vascular Disease in Piglets With Increased Pulmonary Blood Flow

We studied the effect of chronic endothelin A receptor blockade by atrasentan on the pulmonary endothelin-1 system and vascular endothelial growth factor (VEGF) expression in piglets with high pulmonary blood flow. Twenty-five 4-week-old piglets with high pulmonary blood flow were randomized to three groups: sham operated (n = 8), placebo (water) (n = 7), or treatment with atrasentan (2 mg/kg per day) (n = 10). After 3 months, mean pulmonary arterial pressure (PAP) was higher in the placebo group than in the sham group [18 +/- 2 mm Hg versus 14 +/- 1 mm Hg; P < 0.05 (ANOVA)]. Atrasentan treatment was associated with lower cardiac output, PAP (14 +/- 1 mm Hg), and medial wall thickness of pulmonary arteries (diameter: 50-150 microM) compared with placebo [13.6 +/- 3.0% versus 18.1 +/- 4.2%; P < 0.05 (ANOVA)]. Quantitative real-time polymerase chain reaction for endothelin-1, endothelin B receptor, and endothelin-converting enzyme-1 mRNA in lung tissue did not differ. However, immunostaining as well as mRNA for VEGF were lower in atrasentan-treated animals (relative gene expression: atrasentan versus placebo: 0.8 +/- 0.3 versus 1.5 +/- 0.3; P = 0.009). Atrasentan treatment effectively reduces medial hypertrophy in piglets with chronic pulmonary hyperperfusion. Chronic endothelin A receptor blockade by atrasentan may interfere with the expression of VEGF.

Ca2+ Channels and Chronic Hypoxia

Many chronic lung diseases are associated with prolonged exposure to alveolar hypoxia, resulting in the development of pulmonary hypertension. While the exact mechanisms underlying the pathogenesis of hypoxic pulmonary hypertension remain poorly understood, a key role for changes in Ca2+ homeostasis has emerged. Intracellular Ca2+ concentration controls a variety of pulmonary vascular cell functions, including contraction, gene expression, growth, barrier function and synthesis of vasoactive substances. Several studies indicate that prolonged exposure to hypoxia causes alterations in the expression and activity of several Ca2+ handling pathways in pulmonary arterial smooth muscle cells. In contrast, the effect of chronic hypoxia on Ca2+ homeostasis in pulmonary arterial endothelial cells is relatively unexplored. In this review, we discuss data from our laboratory and others describing the effects of prolonged hypoxia on pulmonary vascular smooth muscle and endothelial cell Ca2+ homeostasis and the various Ca2+ channels and handling pathways involved in these responses. We will also highlight future directions of investigation that might improve our understanding of the response of pulmonary vascular cells to chronic hypoxia.

Pulmonary Vascular Endothelial Growth Factor-C in Development and Lung Injury in Preterm Infants

In mice, vascular endothelial growth factor-C (VEGF-C) plays an important role in development of the lymphatic system and in pathogenesis of pulmonary inflammation. Its role in development of the lymphatic system in human lung and in lung injury in newborns remains unclear. We studied the role of VEGF-C in developing human lung, and in acute and chronic lung injury in preterm infants. Included in the immunohistochemistry study were 10 fetuses, 15 control neonates without primary lung disease, 15 preterm infants with respiratory distress syndrome, and 8 infants with bronchopulmonary dysplasia. Tracheal aspirate fluid samples of intubated very-low-birth-weight infants during Postnatal Weeks 1-5 were analyzed with ELISA. Bronchiolar staining for VEGF-C was observed in all 48 samples. Alveolar epithelial staining was seen in most fetuses (8/10). In addition, staining was observed in alveolar macrophages in bronchopulmonary dysplasia (4/8), and late respiratory distress syndrome (2/7). VEGF receptor-3 (VEGFR-3) staining was observed in lymphatic endothelium adjacent to vascular endothelium. VEGF-C was expressed consistently in tracheal aspirate fluid, being highest during the first 2 postnatal days. Antenatal administration of glucocorticoids was associated with higher VEGF-C in tracheal aspirate fluid. The pattern of pulmonary VEGF-C and VEGFR-3 protein expression and consistent VEGF-C protein appearance in tracheal aspirate fluid in human preterm infants indicate a role for VEGF-C in the physiologic development of the lymphatic system of the lung.

Pulmonary hypertension in older children: New approaches and therapies

Pulmonary hypertension contributes significantly to the morbidity and mortality of diverse cardiopulmonary and systemic disorders in children. Advances in vascular biology have led to the identification and characterization of potent endothelium-derived vasoactive mediators, which include nitric oxide (NO), prostacyclin (PgI2) and endothelin-1 (ET-1), that play critical roles in the regulation of pulmonary vascular tone, reactivity, growth and structure. Insights into these agents and their signaling pathways have led directly to the development of current therapies for severe pulmonary hypertension. In this chapter, I will briefly review basic pharmacologic choices for the general management of chronic pulmonary hypertension in older children (outside of the neonatal period). Guidelines and discussion of relevant literature has been recently reviewed.

Treprostinil in Pulmonary Arterial Hypertension

Prostacyclins in PAH - Pathobiological Rationale Endothelial dysfunction is the cornerstone event in the pathobiology of pulmonary arterial hypertension (PAH), a rapidly evolving clinical syndrome of dyspnoea and fatigue eventually leading to right ventricular failure and death.1 In the pulmonary vascular bed, endothelial cells are the major source of mediators modulating pulmonary vascular tone, platelet aggregation and muscular cell growth. These mediators include endothelin-1 (ET-1), nitric oxide (NO) and prostacyclin (PGI2).

Vasculogenesis drives pulmonary vascular growth in the developing chick embryo

Formation of the pulmonary vasculature has been described as occurring by outgrowth of existing vessels (angiogenesis), de novo formation of new vessels (vasculogenesis), or a combination of both processes. Uncertainty about the contribution of angiogenesis and vasculogenesis to pulmonary vascular formation is partly due to methodologic approaches. Evidence in favor of angiogenesis stems from studies that used vascular-filling methods. Such methods identify only directly continuous lumina. Evidence for vasculogenesis has been provided by the use of molecular markers of blood vessel endothelium. Use of both methods has not been combined in the same species, however. We hypothesized, based on published evidence from quail and mouse, that chick pulmonary vascular formation occurs by vasculogenesis. To test that hypothesis, we used vascular filling, serial section, and immunohistochemical methods to analyze the developing lungs of chick embryos from Hamburger and Hamilton stages 20 to 43. Vascular filling suggested that the lumen of the pulmonary arteries sprouted from the sixth pharyngeal arch arteries. However, serial sections and immunohistochemical localization of fetal liver kinase-1 protein, the receptor for vascular endothelial growth factor, showed that the pulmonary arterial tree formed from endothelial cell precursors and coalescence of isolated blood vessels in the mediastinal splanchnic mesenchyme centrally to the developing lung tissue distally. Pulmonary veins grew from the left atrium to the developing lungs. Pulmonary blood vessel formation occurred continuously throughout the embryonic period studied. Our results show that vasculogenesis is the main process by which the pulmonary vasculature forms in the developing chick embryo.

Commentary

HIGHLIGHTED TOPICSLung Growth and RepairCommentaryGary C. SieckGary C. SieckJournal of Applied Physiology October 2004, Volume 97Published Online:01 Oct 2004https://doi.org/10.1152/japplphysiol.00789.2004MoreSectionsPDF (10 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat In the first featured article, entitled “Hypoxia-responsive growth factors upregulate periostin and osteopontin expression via distinct signaling pathways in the rat pulmonary arterial smooth muscle cells,” Dr. P. Li and colleagues (1) examine expression of the extracellular matrix adhesion molecules, periostin and osteopontin, in pulmonary arteries of hypoxia-adapted rats. These investigators also explored the signaling pathways that regulate expression of periostin and osteopontin in response to hypoxia and the hypoxia-responsive growth factors, fibroblast growth factor-1 (FGF-1) and angiotensin II (ANG II). This study is the first to demonstrate that, as a generalized extracellular matrix response to hypoxic stress, the lungs and pulmonary arteries express elevated levels of periostin and osteopontin. Pulmonary arterial smooth muscle cells from hypertrophic pulmonary arteries show localized expression of hypoxia-induced periostin. Coupled with the observation that levels of periostin and osteopontin increase concurrently in lungs, this finding suggests that periostin is involved in extracellular matrix formation and pulmonary vascular hypertrophy and remodeling in response to chronic hypoxic exposure. In vitro studies of pulmonary arterial smooth muscle cells show that growth factor-induced expression of periostin and osteopontin is differentially regulated, suggesting that these adhesion molecules play different roles in pulmonary vascular remodeling under pathophysiological conditions.In the second featured article, entitled “Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice,” Dr. K. Tang and colleagues (3) examine the role of apoptosis in the development of emphysema. These investigators used intratracheal delivery of an adeno-associated cre recombinase virus to downregulate expression of pulmonary vascular endothelial growth factor (VEGF) in adult mice. These investigators found that partial inactivation of pulmonary VEGF leads to alveolar septal cell apoptosis, air space enlargement, and increased lung compliance. Presumably, normal and efficient apoptotic pathways clear the resulting apoptotic cells. However, the lung ineffectively regenerates damaged alveolar walls, and, despite restoration of normal cellular levels of pulmonary VEGF, increased alveolar size and decreased elastic recoil persist. The results of this study suggest that significant loss of VEGF and pulmonary cells, even for only a brief period, leads to long-lasting, possibly permanent effects. It also highlights the protective capacity of VEGF in maintaining lung structure. How long or how often the lung can withstand exposure to damaging insults before repair mechanisms are overwhelmed remains to be elucidated. Loss of structure itself raises the question of whether it is possible to devise pharmacological strategies to replace alveolar septal structures with lung-targeted angiogenesis.In the third and final featured article, entitled “Regional lung growth following pneumonectomy assessed by computed tomography,” Dr. P. Ravikumar and colleagues (2) assessed mechanical strain in remaining lung tissue after pneumonectomy. These investigators quantified lobar volumes and density gradients in both normal and postpneumonectomy lungs to determine whether the remaining lobes expand uniformly and whether tissue densities are changed uniformly. This study is conceptually straightforward and utilizes standard computed tomography technology; however, it illustrates for the first time a powerful and novel use of in vivo imaging to quantify regional lung distortion and changes in local volume, lung compliance, and soft tissue density. These changes can be followed noninvasively and serially in a wide range of clinical and investigational applications that include measuring the extent and progression of regional heterogeneity in lung disease or injury, assessing local response to treatment or surgical intervention, and determining normal or abnormal patterns of lung growth.REFERENCES1 Li P, Oparil S, Feng W, and Chen YF. Hypoxia-responsive growth factors upregulate periostin and osteopontin expression via distinct pathways in rat pulmonary arterial smooth muscle cells. J Appl Physiol 97: 1550–1558, 2004.Google Scholar2 Ravikumar P, Yilmaz C, Dane DM, Johnson RL, Estrera AS, and Hsia CCW. Regional lung growth following pneumonectomy assessed by computed tomography. J Appl Physiol 97: 1567–1574, 2004.Google Scholar3 Tang K, Rossiter HB, Wagner PD, and Breen EC. Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice. J Appl Physiol 97: 1559–1566, 2004.Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 97Issue 4October 2004Pages 1549-1549 Copyright & PermissionsCopyright © 2004 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00789.2004History Published online 1 October 2004 Published in print 1 October 2004 Metrics

Vascular endothelial growth factor expression in metastatic pulmonary tumor from colorectal carcinoma: Utility as a prognostic factor

Objective To define the most reliable prognostic factor, we studied the 5-year survival of patients after resection of pulmonary metastases from colorectal cancer in relation to various prognostic factors, including vascular endothelial growth factor expression in primary and metastatic tumors. Methods A retrospective study was undertaken in 49 patients who had undergone complete resection of pulmonary metastasis from colorectal carcinoma. All patients were retrospectively analyzed for sex, age, location and stage of primary tumor, number of pulmonary metastases, type of pulmonary resection, size of metastatic tumor, lymph node metastasis, and prethoracotomy carcinoembryonic antigen level. Furthermore, vascular endothelial growth factor expression of both primary and metastatic tumors was investigated. Results Overall 5-year survival was 34.3%. In the univariate analysis the number of pulmonary metastases ( P = .007) and vascular endothelial growth factor expression in metastatic tumors ( P = .008) and primary colorectal tumors ( P = .011) were significantly associated with poor survival. In the multivariate analysis the number of pulmonary metastases ( P = .0031), vascular endothelial growth factor expression in metastatic tumors ( P = .0057), and stage of primary tumor ( P = .0321) were characteristics that retained a significant independent prognostic effect on overall survival. A statistically significant difference was not found in the 5-year survival of patients with solitary and negative vascular endothelial growth factor expression in metastatic tumors (59.1%) versus those with multiple and positive vascular endothelial growth factor expression in metastatic tumors (10.0%; P ≤ .0001). Conclusions Vascular endothelial growth factor expression in metastatic pulmonary tumors is an important prognostic marker for patients with pulmonary metastasis from colorectal carcinoma. Our results suggested that patients with multiple and positive vascular endothelial growth factor expression in the metastatic tumor will not receive benefit from metastasectomy.

Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.

Pulmonary vascular endothelial growth factor and nitric oxide interaction during total cardiopulmonary bypass in neonatal pigs

Objective: Lung injury after cardiopulmonary bypass includes pulmonary hypertension and lung edema. Both complications are related to endothelial pulmonary vascular dysfunction, leukocyte sequestration, and increased capillary permeability. This study was done in an attempt to better define the endothelial dysfunction and the cause of edema. Methods: Twenty-five neonatal piglets were subjected to total cardiopulmonary bypass for 90 minutes without crossclamping of the aorta. After weaning from cardiopulmonary bypass, they were allowed to survive 2 hours, at which time they were killed. Preoperative and postoperative hemodynamic studies, lung (n = 16) and muscular (n = 5) vascular endothelial growth factor contents, and exhaled nitric oxide (n = 8) were recorded. Immediately after the animals were killed, pulmonary arterial rings were obtained from 12 piglets and mounted in organ chamber for assessment of endothelial function with receptor-dependent (acetylcholine) or non-receptor-dependent (calcium ionophore A23187) studies and compared with control pulmonary arterial rings. The left lungs of 13 piglets were mounted in isolated perfused lung chambers for filtration coefficient assessment and comparison with control preparations. Results: After cardiopulmonary bypass, pulmonary vascular resistance increased from 953.7 ± 302.6 dyne × s × cm−5 to 1973.6 ± 925.4 dyne × s × cm−5 (P = .03). This was associated with an increase in lung vascular endothelial growth factor content from 91.07 ± 5.314 pg/100 mg tissue to 151.6 ± 11.4 pg/100 mg tissue (P < .0001), an increase in muscle vascular endothelial growth factor from 76.02 ± 11.53 pg/100 mg tissue to 81.58 ± 7.7 pg/100 mg tissue (P not significant), and a decrease in exhaled nitric oxide from 6 ± 1.7 ppb to 3.12 ± 1.4 ppb (P = .003). The filtration coefficient was statistically significantly higher after cardiopulmonary bypass than in control preparations (0.259 ± 0.02 vs 0.525 ± 0.07, P < .0001). Variations in lung vascular endothelial growth factor accumulation were statistically significantly higher than in muscular vascular endothelial growth factor accumulation (60.5 ± 9.1 vs 5.5 ± 5.9, P = .0008). In addition, a statistically significant correlation was found between postbypass lung vascular endothelial growth factor and lung filtration coefficient (P = .0058), as well as between change in lung vascular endothelial growth factor and change in lung filtration coefficient (P = .03). Pulmonary vascular endothelial receptor-dependent (acetylcholine) function was statistically significantly blunted after bypass relative to control values (15.44% ± 4.8% vs 55.5% ± 5.96% of maximal relaxation, P = .0001), whereas non-receptor-dependent endothelial function was unaffected by cardiopulmonary bypass (110.77% ± 8.9% vs 120.63% ± 15.46% of maximal relaxation, P not significant). Conclusions: These findings suggest that lung ischemia that occurs during cardiopulmonary bypass affects the signal transduction from membrane receptors to intracellular calcium mobilization and nitric oxide synthase activation. Lung edema after bypass is probably due in part to lung accumulation of vascular endothelial growth factor, a finding that was not found in systemic muscular nonischemic territories.J Thorac Cardiovasc Surg 2003:125:1050-7

Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure.

To determine whether disruption of vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) signaling in the newborn has long-term effects on lung structure and function, we injected 1-day-old newborn rat pups with a single dose of Su-5416, a VEGFR inhibitor, or vehicle (controls). Lungs from infant (3-wk-old) and adult (3- to 4-mo-old) rats treated with Su-5416 as newborns showed reductions in arterial density (82 and 31%, respectively) and alveolar counts (45 and 29%) compared with controls. Neonatal treatment with Su-5416 increased right ventricle weight to body wt ratios (4.2-fold and 2.0-fold) and pulmonary arterial wall thickness measurements (2.7-fold and 1.6-fold) in infant and adult rats, respectively, indicating marked pulmonary hypertension. We conclude that treatment of newborn rats with the VEGFR inhibitor Su-5416 impaired pulmonary vascular growth and postnatal alveolarization and caused pulmonary hypertension and that these effects were long term, persisting well into adulthood.

Correlations between basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and clinical features of pulmonary arterial hypertension (PAH)

PAH is the result of abnormal pulmonary vascular growth. Elevations in bFGF and VEGF have been noted in animal models, but growth factors that cause PAH in humans are unknown. We measured serum and urine bFGF and VEGF in 76 stable pts with PAH (primary pulmonary hypertension (PPH)=51, collagen vascular disease (CVD)=15, congenital heart disease (CHD)=10), and 10 controls. Growth factors were correlated against age, gender, etiology, NYHA Functional Class, current medications, and hemodynamics.

Serum Levels of Vascular Endothelial Growth Factor and Cavity Formation in Active Pulmonary Tuberculosis

Background: In active pulmonary tuberculosis, certain cytokines have been postulated to be related to cavity formation, although the detailed mechanism of cavity formation is not yet known. Objective: We examined the relationship between cavity formation in pulmonary tuberculosis and vascular endothelial growth factor (VEGF), which functions as an angiogenesis factor. Methods: Forty-eight patients with active pulmonary tuberculosis were divided into two groups according to cavity formation as evaluated by chest high-resolution computed tomography. We evaluated serum VEGF levels by enzyme immunoassay. Results: Group A (with cavities) was comprised of 22 patients and group B (without cavities) was comprised of 26 patients. The serum levels of VEGF were significantly higher in group B (58.733 ± 21.612 pg/ml) than those in normal individuals (8.739 ± 3.656 pg/ml) and in group A (13.053 ± 8.670 pg/ml) (Mann-Whitney U test, p = 0.0149 and p = 0.0481, respectively). Serum levels of interleukin-8 and tumor necrosis factor-α were not significantly different between the two groups. Conclusion: These findings suggested that increased serum VEGF levels subdue cavity formation in active pulmonary tuberculosis.

Pulmonary and systemic vascular tissue collagen, growth factor, and cytokine gene expression in the rabbit

During development, the vascular wall composition of the pulmonary and systemic capacitance vessels and their intravascular pressure changes. Little is known, however, about the factors controlling vascular collagen gene expression in both circulations during growth and development. The purpose of this study was to compare the developmental changes in collagen, major growth factors, and cytokines gene expression, in order to ascertain whether a circulation specific pattern is present in the rabbit. Fetal, neonatal, and adult rabbit extrapulmonary and aortic tissues were obtained and the mRNA levels for collagen I and III, as well as major growth factors and cytokines, were measured by a semi-quantitative RT-PCR technique. Collagen I, but not collagen III, expression was developmentally regulated in pulmonary vascular and aorta tissues. Collagen I expression was greatest during the fetal and neonatal period (P < 0.01) and higher in the aorta as compared with the pulmonary artery at these ages (P < 0.05). Significant developmental changes in growth factor mRNA levels were observed for TGF-beta, IGF-2, and bFGF (P < 0.01). IGF-2 mRNA levels significantly declined in both arteries from neonatal to adult, but bFGF increased only in the pulmonary artery during this transition. With regards to inducible enzymes, COX-2 mRNA levels changed developmentally, whereas iNOS mRNA levels were similar for both vessels at all ages. When comparing the two vessels, COX-2 transcripts were relatively more abundant in the adult pulmonary artery tissue and fetal aorta, with similar levels in the newborn. We conclude that circulation specific developmental regulation of collagen gene expression is present in the rabbit in a pattern that is unrelated to the intravascular pressure.

Pulmonary and systemic vascular tissue collagen, growth factor, and cytokine gene expression in the rabbit

During development, the vascular wall composition of the pulmonary and systemic capacitance vessels and their intravascular pressure changes. Little is known, however, about the factors controlling vascular collagen gene expression in both circulations during growth and development. The purpose of this study was to compare the developmental changes in collagen, major growth factors, and cytokines gene expression, in order to ascertain whether a circulation specific pattern is present in the rabbit. Fetal, neonatal, and adult rabbit extrapulmonary and aortic tissues were obtained and the mRNA levels for collagen I and III, as well as major growth factors and cytokines, were measured by a semi-quantitative RT-PCR technique. Collagen I, but not collagen III, expression was developmentally regulated in pulmonary vascular and aorta tissues. Collagen I expression was greatest during the fetal and neonatal period (P < 0.01) and higher in the aorta as compared with the pulmonary artery at these ages (P < 0.05). Significant developmental changes in growth factor mRNA levels were observed for TGF-beta, IGF-2, and bFGF (P < 0.01). IGF-2 mRNA levels significantly declined in both arteries from neonatal to adult, but bFGF increased only in the pulmonary artery during this transition. With regards to inducible enzymes, COX-2 mRNA levels changed developmentally, whereas iNOS mRNA levels were similar for both vessels at all ages. When comparing the two vessels, COX-2 transcripts were relatively more abundant in the adult pulmonary artery tissue and fetal aorta, with similar levels in the newborn. We conclude that circulation specific developmental regulation of collagen gene expression is present in the rabbit in a pattern that is unrelated to the intravascular pressure.

Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension.

The hypothesis for this study was that increased local expression of vascular angiotensin-converting enzyme (ACE) may contribute to the arterial remodelling which accompanies pulmonary hypertension, since angiotensin II (ANG II) is an important mediator of pulmonary vascular cell growth. The expression of ACE was studied by immunohistochemistry in paraffin-embedded lung sections from adults undergoing heart-lung transplantation for severe primary (n=6) and secondary (n=7) pulmonary arterial hypertension (PH), compared with age-matched controls (n=11). An antigen retrieval technique was used prior to incubating sections with the anti-ACE monoclonal antibody, CG2, or the endothelial marker, monoclonal anti-CD31. In control lungs, the highest level of ACE immunostaining was seen in the alveolar capillary endothelium, with less intense staining in small intra-acinar pulmonary arteries and relatively little staining in larger preacinar arteries. ACE immunostaining was virtually absent in lymphatics and veins. In both primary and secondary PH, there was an increase in ACE immunostaining in the endothelium of intra-acinar peripheral pulmonary arteries compared with control lungs, extending to the level of alveolar ducts, as confirmed by semi-quantitative analysis. The increase in endothelial ACE expression in the intra-acinar arteries of patients with primary and secondary PH is consistent with the hypothesis that locally increased production of ANG II may contribute to the process of pulmonary vascular remodelling.

Increased Pulmonary Blood Flow as a Regulator of Pulmonary Vascular Remodeling: Role of eNOS ♦ 105

To better understand the mechanisms whereby pulmonary blood flow regulates pulmonary vascular bed growth in congenital heart lesions, 42 day old Sprague-Dawley rats (n = 24) had creation of an aortocaval shunt to increase pulmonary blood flow for six weeks. The shunt resulted in a significant increase in heart (2.89 ± 0.4 vs 6.1 ± 0.9, P ≤ 0.05) and lung (1.93 ± 0.5 vs 4.6 ± 1.4, P ≤ 0.05) to body weight (sham vs shunt, mean ± standard deviation) without significantly altering pulmonary (16 ± 3 vs 19 ± 4, NS) or carotid (125 ± 5 vs 130 ± 6, NS) blood pressures (mean mm Hg ± standard deviation). Histologic examination of the lungs revealed significant thickening of the pulmonary arterial medial wall relative to the vessel diameter (35% ± 7.2 vs 57% ± 11.8, P ≤ 0.05, sham vs shunt, mean ± standard deviation) and increased muscularization of very small arteries (≤ 80μM) as evidenced by α-actin smooth muscle staining. Proliferating cell nuclear antigen staining and bromo-deoxyuridine labeling of proliferating cells demonstrated that the thickened arterial wall was a result of medial hypertrophy and not proliferation. To determine the role of nitric oxide (NO) in the regulation of the arteriolar hypertrophy or the affect of hypertrophy on NO expression in the pulmonary vasculature, endothelial NO synthase (eNOS) gene and protein expression were determined in lungs from shunt and sham operated animals. Western and northern analysis demonstrated that eNOS protein and mRNA levels were not altered in the shunt lungs. In addition NAPDH diaphorase staining was negative for increased NOS in the hypertrophied pulmonary vasculature of the shunt animals. Therefore increased pulmonary flow without increased pressure resulted in pulmonary artery medial hypertrophy and not hyperplasia. We speculate that the pulmonary vascular remodeling resulting from increased pulmonary blood flow alone is not mediated by eNOS.

ALTERATIONS IN PULMONARY BLOOD FLOW IN THE LAMB INDUCES CHANGES IN PULMONARY VASCULAR MORPHOLOGY AND GROWTH FACTOR EXPRESSION. • 121

Congenital heart defects are associated with structural abnormalities of the pulmonary circulation, which depend on the amount of pulmonary blood flow. With increased blood flow, there is increased pulmonary artery size, medial smooth muscle hypertrophy, and extension of this muscle into nonmuscular pulmonary arteries. With decreased blood flow, there is decreased number and size of pulmonary arteries, and decreased muscularity. We have developed models of increased (insertion of an aortopulmonary vascular graft) and decreased (unilateral pulmonary artery banding) pulmonary blood flow in fetal lambs. By 4 weeks of age, these lambs demonstrate structural abnormalities of the pulmonary circulation. The cellular and molecular mechanisms underlying these changes are not understood. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have effects on the proliferation of endothelial and vascular smooth muscle cells, and on the formation of new capillaries and larger vessels. To evaluate the role of these growth factors in controlling pulmonary vascular growth, we cloned ovine bFGF and VEGF cDNA fragments. Lung tissues were obtained from 4-week-old lambs with normal, increased or decreased pulmonary blood flow. RNase protection assays,in situ hybridization, and Western blot analyses (using commercial antibodies) were performed. Increased pulmonary blood flow, with resulting increased size and number of pulmonary arteries, is associated with increased gene and protein expression for bFGF (3-fold) and VEGF (5-fold); while decreased pulmonary blood flow, with resulting decreased size and number of pulmonary arteries, is associated with decreased gene and protein expression for bFGF (2-fold) and VEGF (2-fold). A better understanding of the cellular and molecular mechanisms controlling pulmonary vascular growth may lead to new treatment strategies for children with congenital heart defects.

Neutral Endopeptidase-24.11 Inhibition Reduces Pulmonary Hypertension and Pulmonary Vascular Growth (Remodelling) in the Rat

Neutral Endopeptidase-24.11 Inhibition Reduces Pulmonary Hypertension and Pulmonary Vascular Growth (Remodelling) in the Rat