Pulmonary Vascular Endothelium Research Articles

L-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets

Newborn piglets develop pulmonary hypertension and have diminished pulmonary vascular nitric oxide (NO) production when exposed to chronic hypoxia. NO is produced by endothelial NO synthase (eNOS) in the pulmonary vascular endothelium using l-arginine as a substrate and producing l-citrulline as a byproduct. l-Citrulline is metabolized to l-arginine by two enzymes that are colocated with eNOS in pulmonary vascular endothelial cells. The purpose of this study was to determine whether oral supplementation with l-citrulline during exposure of newborn piglets to 10 days of chronic hypoxia would prevent the development of pulmonary hypertension and increase pulmonary NO production. A total of 17 hypoxic and 17 normoxic control piglets were studied. Six of the 17 hypoxic piglets were supplemented with oral l-citrulline starting on the first day of hypoxia. l-Citrulline supplementation was provided orally twice a day. After 10 days of hypoxia or normoxia, the animals were anesthetized, hemodynamic measurements were performed, and the lungs were perfused in situ. Pulmonary arterial pressure and pulmonary vascular resistance were significantly lower in hypoxic animals treated with l-citrulline compared with untreated hypoxic animals (P < 0.001). In vivo exhaled NO production (P = 0.03) and nitrite/nitrate accumulation in the perfusate of isolated lungs (P = 0.04) were significantly higher in l-citrulline-treated hypoxic animals compared with untreated hypoxic animals. l-Citrulline supplementation ameliorated the development of pulmonary hypertension and increased NO production in piglets exposed to chronic hypoxia. We speculate that l-citrulline may benefit neonates exposed to prolonged periods of hypoxia from cardiac or pulmonary causes.

Ca2+ entry via α1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

Phosphodiesterase Type 5 Inhibition

The development of right heart failure has been a clinical concern of physicians caring for patients after cardiac transplantation since the inception of the field.1 Although the problem of “fixed” pulmonary vasoconstriction that does not respond to the reduction of left ventricular filling pressure seems to have diminished since the early days of transplantation, there are still some patients in whom this remains a problem. Data from the International Society of Heart Transplantation Registry suggests that right ventricular (RV) dysfunction accounted for 50% of all cardiac complications and 19% of all early deaths among heart transplantation patients,2 with persistent pulmonary vasoconstriction a major contributing factor to early RV dysfunction. The cause of the increase in vasoconstriction is unknown, although it may be a result of humoral factors, including cytokines, promoting changes in vascular smooth muscle gene expression. The hallmark of this problem is a lack of sensitivity to endogenous and exogenous vasodilators, be they nitric oxide (NO) released from pulmonary vascular endothelium or exogenous agents acting through NO, endothelin, or prostaglandin signaling pathways.3 Heart failure patients with fixed pulmonary hypertension can be disqualified from consideration for transplant surgery, given their worse prognosis for early survival. One alternative for these patients is to attempt …

Blockade of leucocyte function-associated antigen-1 (LFA-1) decreases lymphocyte trapping in the normal pulmonary vasculature: studies in the isolated buffer-perfused rat lung.

Adhesion molecules regulate the migration of lymphocytes in lymphoid and non-lymphoid organs. In the lung, little is known about lymphocyte sticking and migration through the pulmonary vascular endothelium in physiological or pathological situations. Therefore the isolated buffer-perfused rat lung was used to investigate the mobilization of lymphocytes out of the normal lung into the venous effluent and to the bronchoalveolar space. The lymphocyte subset composition was characterized in the venous effluent, the lung tissue and the bronchoalveolar lavage (BAL) using immunocytology. Lymphocytes continuously left the normal lung at a total of 5.0 +/- 0.7 x 106 cells within the first hour of perfusion. The injection of 200 x 106 lymphocytes via the pulmonary trunk increased the venous release of lymphocytes by 170%. To investigate the effect of LFA-1 and CD44 on the adhesion of lymphocytes to the pulmonary endothelium, lymphocytes preincubated with an anti-LFA-1 MoAb, which blocks the interaction of LFA-1 and intercellular adhesion molecule-1 (ICAM-1), or lymphocytes preincubated with an anti-CD44 MoAb, were injected. The injection of LFA-1-blocked lymphocytes led to an increase by 70% of injected cells recovered in the perfusate within the first hour, whereas anti-CD44 treatment of injected lymphocytes had no effect. The LFA-1-blocked lymphocytes showed higher numbers of T and B cells in the effluent. Thus, the present experiments demonstrate that LFA-1 influences the trapping of lymphocytes in the vasculature of the healthy rat lung.

Dysfunction of Pulmonary Vascular Endothelium in Chronic Obstructive Pulmonary Disease: Basic Considerations for Future Drug Development

Chronic obstructive pulmonary disease (COPD) is one of the leading health problems worldwide and continues to be a major cause of morbidity and mortality in developed countries. The clinical features of COPD are chronic obstructive bronchiolitis and emphysema. Pulmonary vascular endothelial dysfunction is a characteristic pathological finding of COPD at different stages of the disease. Functional changes of pulmonary endothelial cells in COPD include antiplatelet abnormalities, anticoagulant disturbances, endothelial activation, atherogenesis, and compromised regulation of vascular tone which may adversely affect the ventilation-perfusion match in COPD. As the most important risk factor of COPD, cigarette smoking may initiate pulmonary vascular impairment through direct injury of endothelial cells or release of inflammatory mediators. Morphological changes such as denudation of endothelium and endothelial cell apoptosis have been observed in the pulmonary vasculature in COPD patients as well as functional alterations. Changes in the expression of tissue factor pathway inhibitor (TFPI), thrombomodulin, selectins, and adhesion molecules in pulmonary endothelial cells as well as complex regulation and interaction of vasoactive substances and growth factors released from endothelium may underlie the mechanisms of pulmonary endothelial dysfunction in COPD. The mechanism of endothelial repair/regeneration in COPD, although not fully understood, may involve upregulation of vascular endothelial growth factors in the early stages along with an increased number of bone marrow-derived progenitor cells. These factors should be taken into account when developing new strategies for the pharmacological therapy of patients with COPD.

Changes of blood coagulative and fibrinolytic system and function of pulmonary vascular endothelium after therapy in patients with acute pulmonary thromboembolism

To study the changes of blood coagulative and fibrinolytic system and the function of pulmonary vascular endothelium in the course of acute pulmonary thromboembolism (PTE) and after anticoagulant or thrombolytic treatment. Twenty patients with acute non-massive PTE, 10 males and 10 females, aged (57 +/- 11) underwent anticoagulant treatment and 17 sex-, and age-matched acute massive PTE patients underwent thrombolytic treatment. The plasma level of D-dimer (D-D), thrombomodulin (TM), protein C (PC), protein S (PS), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and antithrombin-III (AT-III) activity were measured by ELISA before and after normal subjects severed as control group were included in the study. The plasma level of D-D, PS, PC, TM, t-PA and PAI-1 were measured by a method of ELISA before the treatment and six days after the anticoagulant treatment or 24 hours after the thrombolytic treatment. AT-III activity was measured by chromo-substrate method at the same time points. Forty sex- and age-matched healthy persons were used as controls. The plasma levels of D-D, t-PA, PS, and TM level of the anticoagulant group were all significantly higher and the AT-III activity of the 2 treatment groups was significantly lower than those of the controls before treatment (all P < 0.05); the plasma levels of D-D, t-PA, PAI-1, PS, and TM of the thrombolytic group were ala significantly higher and the AT-III activity was significantly lower than those of the control group before the treatment (all P < 0.05). After anticoagulant therapy, the plasma levels of D-D, t-PA, PS, and PC were significantly lower than those before treatment (all P < 0.05), however, the plasma levels of PAI-1, TM, and AT-III activity after treatment did not differ significantly from those before treatment. The plasma levels of D-D, t-PA, PS, PC, and TM after treatment of the thrombolytic group were all significantly lower than those before treatment (all P < 0.05), however, the plasma levels of PAI-1, TM, and AT-III activity after treatment did not differ significantly from those before treatment. Apparent imbalance in the blood coagulative and fibrinolytic system and pulmonary vascular endothelium damage occur in the patients with acute PTE. Combination tests of plasma D-D, AT-III, PS, PC, TM, t-PA and PAI-1 can give a more comprehensive explanation of the imbalance in the blood coagulative and fibrolytic system. Anticoagulant treatment and thrombolytic treatment play important roles in the regulation of the imbalance of coagulative and fibrinolytic system and protection of the function of pulmonary vascular endothelium of PTE patients.

Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.

Comparison of relaxation responses to multiple vasodilators in TxA2‐analog and endothelin‐1‐precontracted pulmonary arteries

Peri-operative pulmonary hypertension can lead to right ventricular dysfunction and to an increase in morbidity and mortality. Altered function of the pulmonary vascular endothelium and vasoconstriction play a crucial role in the development of elevated pulmonary vascular resistance. Because pulmonary artery vasoreactivity is dependent on many factors including the constricting agent that precipitated the event therefore the aim of the current study was to investigate the effectiveness of different classes of vasodilator agents to reverse endothelin-1 (ET-1) or thromboxane A(2) (TxA(2))-induced vasoconstriction in porcine pulmonary artery (PA) in vitro. Relaxation responses to vasodilatory drugs were studied in PA precontracted with ET-1 (1 x 10(-8) M) or TxA(2) analog (U46619, 1 x 10(-8) M). All vasodilating drugs were added in a cumulative fashion and isometric tension measurements were obtained using an organ chamber technique. In both groups relaxation responses to the vasodilators were dose dependent. When ET-1 was used as a constrictor nitroglycerin and milrinone caused nearly complete (80-100%) relaxation, whereas other agents were of limited effectiveness (40-50%). On the other hand, in the vessels constricted with U46619, olprinone, indomethacin, prostaglandin E(1) (PGE(1)), nitroglycerin, milrinone and clevidipine induced complete (90-110%) vasodilatation but brain natriuretic peptide (BNP), L-arginine, and isoproterenol relaxed the vessels maximally by 45-60%. Nitroglycerin and milrinone are very effective in reversing ET-1 and U46619-induced pulmonary vasoconstriction in vitro. The effectiveness of other drugs studied was dependent on the type of constrictor used. BNP, L-arginine and isoproterenol were shown to have minimal vasodilatory effects in porcine PA.

REBUTTAL FROM DRS. ROCHEFORT AND MICHELAKIS

POINT-COUNTERPOINTREBUTTAL FROM DRS. ROCHEFORT AND MICHELAKISPublished Online:01 May 2007https://doi.org/10.1152/japplphysiol.01258c.2006MoreSectionsPDF (39 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Opening his argument, our opponent calls you, the readers, to a gambling exercise; admittedly a safe bet. Because in one of his reviews on the subject, only four papers found the endothelium to be nonessential for HPV, compared to the 15 that found it to be essential (1), he asks you to bet your money on his side of the argument. However, even in Las Vegas you get in trouble if you play safe bets, but with weighted dice! To support their side of the story in that review, the authors listed (in Table 1 of that review) apparently all the papers that were studying sustained HPV (more than 20 min of hypoxia) (1). We do not understand the basis on which 20 min was used as the cutoff between acute and “sustained HPV.” But, even using this rule, several papers were omitted from that table and argument, which, interestingly, strongly suggest a nonessential role of the endothelium. For example, Wang et al. (3) studied isolated lamb pulmonary arteries: even up to 120 min of hypoxia, caused the same constriction in the vessels with and without endothelium. In fact, the endothelium-denuded vessels had a faster constriction to hypoxia. Jane Madden's work [including the paper that we discussed in our argument and in our Fig. 1A (2)] is also not included in the 4/15 argument. We could improve the odds more by adding papers from our group (published before and after that review), but lucky you, you are scientists and not gamblers.Nevertheless, this Point:Counterpoint series proves to be much easier to settle than expected. For example, our opponent alerts you to the fact that we would try to downgrade the importance of endothelial-derived factors by presenting them as modulators of HPV. But we do not really have to. He has done this for us, in the “gambler's guide to HPV,” that he offered you (2). The conclusion of this review reads: “mediators derived from the pulmonary vascular endothelium exert a powerful modulating influence on the response of the pulmonary circulation to hypoxia.” We could not agree more.In his current argument, our opponent concludes: “It is, therefore, possible that the activation of Rho kinase during sustained HPV represents the bridge between physiological HPV… and the pathophysiological consequences of chronic hypoxia.”REFERENCES1 Aaronson PI, Robertson TP, Ward JP. Endothelium-derived mediators and hypoxic pulmonary vasoconstriction. Respir Physiol Neurobiol 132: 107–120, 2002.Crossref | PubMed | ISI | Google Scholar2 Madden JA, Vadula MS, Kurup VP. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 263: L384–L393, 1992.Abstract | ISI | Google Scholar3 Wang Y, Coe Y, Toyoda O, Coceani F. Involvement of endothelin-1 in hypoxic pulmonary vasoconstriction in the lamb. J Physiol 482: 421–434, 1995.Crossref | PubMed | ISI | Google Scholar Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 102Issue 5May 2007Pages 2075a-2076 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.01258c.2006History Published online 1 May 2007 Published in print 1 May 2007 PDF download Metrics Downloaded 79 times

Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary hypertension

Idiopathic pulmonary arterial hypertension (PAH) is characterized by proliferation of pulmonary vascular endothelial and smooth muscle cells causing increased vascular resistance and right heart failure. Mutations in the bone morphogenetic protein receptor type 2 (BMPR2) are believed to cause the familial form of the disease. Reduced expression of BMPR2 is also noted in secondary PAH. Recent advances in the therapy of PAH have improved quality of life and survival, but many patients continue to do poorly. The possibility of treating PAH via improving BMPR2 signaling is thus a rational consideration. Such an approach could be synergistic with or additive to current treatments. We developed adenoviral vectors containing the BMPR2 gene. Transfection of cells in vitro resulted in upregulation of SMAD signaling and reduced cell proliferation. Targeted delivery of vector to the pulmonary vascular endothelium of rats substantially reduced the pulmonary hypertensive response to chronic hypoxia, as reflected by reductions in pulmonary artery and right ventricular pressures, right ventricular hypertrophy, and muscularization of distal pulmonary arterioles. These data provide further evidence for a role for BMPR2 in PAH and provide a rationale for the development of therapies aimed at improving BMPR2 signaling.

Effects of Phosphodiesterase 5 Inhibitor on Pulmonary Vascular Reactivity in the Fetal Lamb

Nitric oxide released by pulmonary vascular endothelium is a potent vasodilator related to increased cyclic guanosine monophosphate (cGMP) content. Hydrolysis of cGMP is achieved predominately by cGMP-specific phosphodiesterases. Sildenafil is a selective phosphodiesterase-5 (PDE5) inhibitor. The purpose of the study is to assess the effects of sildenafil on pulmonary vascular circulation during the perinatal period. Thirty-two pregnant ewes were operated on at the end of gestation, and fetal lambs were prepared with catheters placed into the aorta, vena cava, pulmonary artery, and left atrium. An ultrasonic flow transducer and an inflatable vascular occluder were placed respectively around the left pulmonary artery and the ductus arteriosus. Fetal lambs were randomly divided into two groups: (1) sildenafil group, infused continuously with sildenafil for 24 hours at a rate of 1 mg/h; or (2) control group, infused with saline for 24 hours. After 24 hours of infusion, we compared basal pulmonary vascular resistance and the pulmonary vascular responses to increase in fetal PaO2 and to acute ductus arteriosus compression causing "shear stress." Sildenafil infusion did not change mean aortic and pulmonary artery pressures, increased mean left pulmonary blood flow by 160%, and decreased pulmonary vascular resistance by 60% (p < 0.05). However, both mean flow (Q) and pulmonary vascular resistance returned to baseline values after 2 hours of sildenafil infusion. Despite similar baseline values, pulmonary vascular resistance during maternal O2 inhalation was lower in the sildenafil group than in the control group (0.21 +/- 0.03 versus 0.33 +/- 0.03 mm Hg.mL(-1).min(-1), respectively; p < 0.01). Furthermore, drop in pulmonary vascular resistance during acute ductus arteriosus compression was greater in the sildenafil group (from 0.56 +/- 0.06 to 0.26 +/- 0.04 mm Hg.mL(-1).min(-1)) than in the control group (from 0.55 +/- 0.05 to 0.39 +/- 0.03 mm Hg.mL(-1).min(-1); p < 0.01). Although sildenafil induces a transient pulmonary vasodilation, it mediates a sustained change in vascular reactivity, especially to birth-related stimuli in the ovine fetal lung. These data suggest that PDE5 is involved in the regulation of pulmonary vascular reactivity during the perinatal period and may potentiate birth-related pulmonary vasodilator stimuli.

PREVENTION OF ISCHEMIA REPERFUSION INJURY BY POSITIVE PULMONARY VENOUS PRESSURE IN ISOLATED RAT LUNG

Pulmonary ischemia-reperfusion (I/R) without tissue hypoxia induces inflammatory cytokine mRNA expression in the lung under the condition of 0 mm Hg pulmonary venous pressure (0PVP), which might be a cause of I/R injury. Our aim is to determine whether the pulmonary vascular endothelium expresses cytokine mRNAs and their corresponding proteins or develops I/R injury when positive PVP is maintained during ischemia to provide a positive stretch to the endothelium throughout the ischemic period. In isolated, perfused, and ventilated rat lungs, the right and left pulmonary arteries were isolated, and the left lung was selectively occluded for 60 min and then reperfused for 30 min. During ischemia, the left atrial pressure was maintained at 5 mm Hg (5PVP) or 0PVP. TNF-alpha, IL-1beta, IL-6, and IL-10 mRNA expression in the lungs was evaluated by RT-PCR and in situ hybridization, and the production and localization of corresponding proteins were determined by staining with fluorescence-labeled antibodies against the cytokines and an antibody against CD34. Pulmonary vascular/epithelial permeability was evaluated by measuring albumin content in bronchoalveolar lavage (BAL) fluid and wet/dry ratio. At 5PVP, there were no increases in the left lung perfusion pressure, albumin content in BAL fluid, wet/dry ratio, or expression of cytokine mRNAs and their corresponding proteins on the vascular endothelium by I/R. In contrast, at 0PVP, the increased expression of cytokine mRNAs and their corresponding proteins on the vascular endothelium by I/R was verified. The finding that the application of 5PVP during ischemia abolished the expression of cytokine mRNAs and their corresponding proteins as well as the I/R injury gives us new insights in the study of lung preservation for transplantation.

Effects of the very late adhesion molecule 4 antagonist WAY103 on human peripheral blood eosinophil vascular cell adhesion molecule 1–dependent functions

Eosinophil infiltration to the lung in allergic inflammation can be initiated by the tethering of circulating cells through very late adhesion molecule 4 (VLA-4; alpha4beta1, CD49d/CD29) to vascular cell adhesion molecule 1 (VCAM-1) expressed on pulmonary vascular endothelium. Small-molecule VLA-4 antagonists have been proposed as a therapeutic mechanism to prevent eosinophil infiltration in asthma; however, they might affect other eosinophil functions. The small-molecule VLA-4 antagonist (2S)-3-(4-Dimethylcarbamoyloxyphenyl)-2-{[(4R)-5,5-dimethyl-3-(1-methyl-1H-pyrazole-4 sulfonyl)thiazolidine-4-carbonyl]amino}propionic acid (WAY103) was assessed for its effects on eosinophil VLA-4-dependent functions, including adhesion, migration, respiratory burst, and degranulation. Human peripheral blood eosinophils were preincubated with WAY103, anti-alpha4, and/or anti-beta2 integrin mAbs and then assessed for adhesion to recombinant VCAM-1, intercellular adhesion molecule 1, and endothelial cell monolayers. Transmigration was measured by using human pulmonary microvascular endothelial cell monolayers and Transwell filters. Superoxide anion generation was determined by means of cytochrome C reduction and degranulation by means of eosinophil-derived neurotoxin release. WAY103 inhibition of eosinophil adhesion to recombinant VCAM-1 was dose dependent (63% inhibition with 100 nM WAY103, P < .04) and comparable with inhibition caused by anti-alpha4 mAb (60.1% inhibition). Although pretreatment with WAY103 also decreased eosinophil adhesion to TNF-alpha plus IL-4-activated human pulmonary microvascular endothelial cell monolayers, it did not prevent eosinophil transendothelial migration in response to RANTES. Finally, WAY103 inhibited VCAM-1-stimulated superoxide generation but enhanced cytokine-activated eosinophil-derived neurotoxin degranulation. Although small-molecule VLA-4 antagonists, such as WAY103, might reduce eosinophil adhesion, this approach might not be sufficient to eliminate this cell from in vivo allergic airway inflammatory participation and could even promote specific cell activation.

Objective and noninvasive detection of sub-clinical lung injury in breast cancer patients after radiotherapy

Aims We employed technetium-99m hexamethylpropylene amine oxime (Tc-99m HMPAO) lung scan to detect sub-clinical lung injury after radiation therapy of 60 female patients diagnosed with right breast cancer. Methods The degree of pulmonary vascular endothelium damage was represented as lung/liver uptake ratios (L/L ratios) calculated on Tc-99m HMPAO lung scan. All patients underwent simple mastectomy and post-operative radiotherapy of approximately 50 Gy. We divided the patients into three groups according to the interval between radiotherapy and lung Tc-99m HMPAO lung scan: Group 1 included 20 patients who received the lung scan within 1–3 months after radiotherapy, group 2 included 20 patients were within 3–9 months after radiotherapy, and group 3 included 20 patients were more than 9 months after radiotherapy. In addition, 20 age-matched normal women were included as the control group. Results The L/L ratios were 0.32±0.04 for normal controls, 0.59±0.10 for group 1, 0.55±0.07 for group 2, and 0.34±0.04 for group 3, respectively. Based on our preliminary results, we found that sub-clinical lung injury and significantly increased L/L ratio in breast cancer patients received radiotherapy may occur within the first 6 months after radiotherapy. However, the L/L ratio is markedly decreased after 9 months. Conclusion Our findings concluded that the degree of pulmonary vascular endothelium damage represented as the L/L ratio on Tc-99m HMPAO lung scan has the potential to be a sensitive, objective and noninvasive method to detect sub-clinical lung injury in breast cancer patients received radiotherapy.

Bone Morphogenetic Protein Receptor-2 and Pulmonary Arterial Hypertension

See related article, pages 1053–1063 Pulmonary arterial hypertension (PAH) is an intriguing condition that is characterized by dramatic changes in the structure and function of pulmonary microcirculation, particularly at the level of the distal arteriolar bed. In addition to exuberant hypertrophy of the medial smooth muscle layers, the pathological features of PAH include hypertrophy and fibrosis of the intima, and occasionally the appearance of plexiform lesions which often occur distal to regions of arterial occlusion.1 Plexiform lesions have attracted considerable attention even though these are not necessarily present in all patients with primary or idiopathic PAH, and they are also found in patients with PAH associated with known causes such as congenital heart disease, collagen vascular disease, and so forth. Hyperproliferative endothelial cells (EC) represent an important component of these complex glomeruloid structures, and have in some cases been shown to be of monoclonal origin.2 Interestingly, although increased smooth muscle cell (SMC) and EC growth may predominate in the advanced stages of disease, more recent evidence suggest that increased apoptosis and loss of pulmonary vascular endothelium may predominate at earlier stages. Indeed selection pressure created by profound EC loss may create the conditions necessary for the emergence of apoptosis resistant and hyperproliferative endothelial cell “clones.”3 The discovery of heterozygous mutations of the BMPR2 gene, encoding for the bone morphogenetic protein receptor-II (BMPR-II), in a substantial proportion of patients with familial pulmonary arterial hypertension (FPAH), as well as many cases of sporadic or idiopathic disease (IPAH),4–6 represents perhaps the single greatest advance toward an understanding of the molecular mechanisms that underpin this puzzling and often lethal vascular disease. However, after the initial excitement of discovery, we are now left with a number of challenging questions related to how loss-of-function mutations scattered throughout the 13 exons of …

811. ACE-Targeted eNOS and BMPR2 Gene Therapy Attenuates Pulmonary Hypertension in a Chronic Hypoxia Rat Model

Idiopathic pulmonary arterial hypertension is a fatal disease characterized by vascular remodeling and right ventricular failure. Modern vasodilator therapies improve prognosis but ultimately most patients succumb or require heart-lung transplantation. We previously developed targeted adenoviral (Ad) strategies for pulmonary vascular gene delivery based on the use of a bispecific conjugate (Fab-9B9) that targets Ad infection to angiotensin converting enzyme (ACE) expressed on pulmonary vascular endothelium. In the current study we determined whether targeting achieved therapeutic gains when using Ad gene delivery of endothelial nitric oxide synthase (eNOS), then evaluated a more novel approach using targeted delivery of the gene for bone morphogenetic protein receptor type 2, based on the knowledge that mutations in this receptor and defects in BMPR2 signaling account for many of the inherited forms of IPAH. Rats (n=6) were injected with 5|[times]|109 pfu of either irrelevant virus, AdCMVeNOS, AdCMVeNOS+Fab9B9 or saline (PBS), then exposed to 10% oxygen atmosphere for three weeks, then right ventricular systolic pressure (RVSP), mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (Fulton Index, FI) were determined. A concurrent group of rats maintained in normoxic conditions was used as a control. Hypoxia lead to increased RVSP (42.2|[plusmn]|4.6mmHg), PAP (34.5|[plusmn]|2.8 mmHg) and FI (0.48|[plusmn]|0.02) vs normoxic controls (22.3|[plusmn]|0.8, 19.4|[plusmn]|1.2 and 0.26|[plusmn]|0.01 respectively). AdCMVeNOS attenuated RVSP(33.9|[plusmn]|1.9), PAP(27|[plusmn]|2.3) and FI(0.41|[plusmn]|.03), with a further therapeutic gain seen with Fab-9B9 targeting (RVSP 28.0|[plusmn]|1.8). A separate experiment using irrelevant virus+Fab-9B9, AdCMVBMPR2+Fab-9B9 or PBS showed that BMPR2 gene delivery attenuated the hypoxia-induced increase in RVSP, PAP and FI by 27%, 34% and 50% respectively. Irrelevant Ad alone or with Fab-9B9 had no effect. Thus, the novel findings are that Ad-mediated eNOS delivery attenuates chronic hypoxia-induced PHT, and this effect is improved with targeting. This is also the first demonstration that upregulation of BMPR2 signaling attenuates pulmonary hypertension, suggesting a therapeutic strategy for patients with BMPR2 mutations.

Receptor Protein Tyrosine Phosphatase μ Regulates the Paracellular Pathway in Human Lung Microvascular Endothelia

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

Noninvasive assessment and monitoring of the pulmonary circulation

In pulmonary vascular disease, changes in the pulmonary vascular bed will lead to altered pulmonary haemodynamics. This review describes the application of several physiological principles to measure these changes noninvasively by means of novel techniques. Flow characteristics of blood through the pulmonary vascular bed alter in pulmonary vascular disease. Recent developments in magnetic resonance imaging and computed tomography make it possible to visualise and quantify these abnormal flow patterns. Information regarding pulmonary perfusion can also be obtained by measuring the electrical impedance changes in the lung by electrical impedance tomography. A more indirect approach to measure the pulmonary blood flow is the measurement of the absorption of acetylene, a perfusion limited gas. Information on the pulmonary vascular bed can also be obtained by the measurement of exhaled products of the pulmonary vascular endothelium, such as nitric oxide. Although all the techniques described offer new ways to diagnose or monitor pulmonary vascular disease, clinical data on these techniques are limited. Further improvement and evaluation of the clinical value of these techniques are therefore obligatory before they can be used in clinical practice.

Endothelial cells from bovine pulmonary microvasculature respond to Mycoplasma bovis preferentially with signals for mononuclear cell transmigration

Mycoplasma bovis can cause arthritis or mastitis following pneumonia and mycoplasmemia in cattle. Interactions with pulmonary vascular endothelium have been recorded as localized vasculitis, perivascular mononuclear cell infiltrations, and accumulation of inflammatory cells in lesions. We compared adhesion mediators and cytokine gene expression as well as cytotoxicity of cultured primary bovine aortic and bovine pulmonary microvascular endothelial cells (BPMEC) challenged with M. bovis. We also tested if abscess-forming ability of strains of M. bovis is associated with changes on endothelial cells. Increased VCAM-1 surface expression was found in both cell types, while only infected BPMEC increased MCP-1 transcription, both mediators specific for mononuclear cell transmigration. Given no induction of ICAM-1 mRNA in either cell type, induction of IL-8 mRNA by BPMEC suggested that neutrophil transmigration was signaled in microvascular areas. Infected BPMEC showed early induction of IL-1β and IL-6 mRNA. Excepting VCAM-1, differential strain effects were limited to BPMEC and not correlated with their abscess-forming capability. In addition, only strain DSA16 had minor cytotoxic effect on both cell types. We thus show that BPMEC are more susceptible than aortic cells to M. bovis-induced activation. Activation preferentially yielded signals for mononuclear cell transmigration, correlating well with in vivo observations of infiltrating cells at pulmonary sites.

173 Effects of Phosphodiesterase 5 Inhibitor on Pulmonary Vascular Reactivity in The Fetal Lamb

Background: Nitric Oxide (NO) released by pulmonary vascular endothelium is a potent vasodilator related to increased cGMP content. Hydrolysis of cGMP is achieved predominantly by cGMP–specific phosphodiesterases (PDEs). Sildenafil (SILD) is a selective phosphodiesterase–5 (PDE–5) inhibitor. The purpose of the study was to assess the effects of sildenafil on pulmonary vascular circulation during the perinatal period.Methods: 12 pregnant ewes were operated on between 128 and 130 days gestation (term = 145 d). Catheters were placed into the ascending aorta, superior vena cava, main pulmonary artery and left atrium. An ultrasonic flow transducer was placed around left pulmonary artery (LPA). An inflatable vascular occluder was placed around ductus arteriosus (DA). Acute DA compression increases pulmonary artery pressure (PAP) causing an increase in pulmonary blood flow (Q) and a progressive decline in pulmonary vascular resistance (PVR). Fetal lambs were divided in two groups: 1) a SILD group infused continuously with SILD at a rate of 24 mg/day (n=6) and; 2/ control group (CONT) infused with saline (n=6). After 24 hrs of infusion, we compared the hemodynamic response of LPA to: 1) increased fetal PaO2 (maternal O2 inhalation for 30 min), and; 2) increase in vascular shear-stress (flow-induced pulmonary vasodilatation related to DA compression).Results: Mean PAP, Q and PVR were similar in both groups before and after 24 h of SILD or saline infusion. Despite similar baseline values, PVR during maternal O2 inhalation was lower in SILD than in CONT group (0.23±0.01 vs 0.27±0.01 mmHg/ml.min−1 respectively) (p<0.01). Furthermore, drop in PVR during acute DA compression was greater in the SILD group (from 0.54±0.03 to 0.26±0.02 mmHg/ml.min−1) than in the CONT group (from 0.55±0.04 to 0.39±0.02 mmHg/ml.min−1) (p<0.01).Conclusion: Although no difference was found in the basal pulmonary vascular tone, sildenafil increases pulmonary vascular reactivity in the ovine fetus. These data suggest that PDE–5 is involved in the regulation of pulmonary vascular reactivity during the perinatal period. We further speculate that specific inhibitor of PDE–5 may potentiate birth related pulmonary vasodilator stimuli and could improve conditions associated with failure to circulatory adaptation at birth.