Ligation Of Left Pulmonary Artery Research Articles

Development and Characterization of a Novel Rat Model for Emulating COPD-Associated Cor Pulmonale.

Cor pulmonale, a condition marked by right ventricular (RV) dysfunction, is frequently associated with chronic obstructive pulmonary disease (COPD) and significantly worsens COPD prognosis. Despite its clinical relevance, the development of effective treatments is hindered by the lack of animal models that accurately replicate the complex interplay between COPD and cor pulmonale. This study introduces a novel rat model combining cigarette smoke (CS) exposure with left pulmonary artery ligation (LPAL) to better mimic the pathophysiological features of COPD-cor pulmonale. Pulmonary function tests revealed impaired lung function, and histological assessments indicated emphysematous changes and inflammatory infiltration, consistent with COPD pathology. Furthermore, the model exhibited hallmarks of cor pulmonale, including right ventricular hypertrophy, fibrosis, and capillary rarefaction, alongside hemodynamic alterations indicative of pulmonary hypertension. This study's findings underscore the potential of the LPAL+CS rat model to advance understanding of COPD-cor pulmonale pathophysiology and facilitate the development of targeted therapeutics.

Establishment of mouse models for severe pulmonary hypertension through 'double-hit' strategies.

Mouse models are crucial for understanding pulmonary hypertension (PH) mechanisms and developing therapies, but existing mouse models under hypoxia only exhibit mild PH. To address this, we established a double-hit model combining unilateral pneumonectomy (LPx) or left pulmonary artery ligation (LPAL) with hypoxia exposure in C57BL/6 mice. Our detailed haemodynamic and histological evaluations post-surgery demonstrated pronounced elevations in right ventricular systolic pressure (RVSP) (LPAL: 41.1±4.63mmHg, P=0.005; LPx: 38.4±2.95mmHg, P=0.002; Sham: 32.1±2.21mmHg) and pulmonary vascular wall thickness (LPAL: 56.9±3.34%, P=0.02; LPx: 54.3±4.65%, P=0.04; Sham: 44.8±3.76%) compared to hypoxia-exposed sham-operated controls, reflecting a more severe PH phenotype. These novel models, which exhibit haemodynamic alterations akin to the established hypoxia with SU5416-induced PH model as per published data, could offer a substantial contribution to future PH research and therapeutic development.

A Dynamic Sheep Model to Induce Pulmonary Hypertension and Right Ventricular Failure.

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript describes the method to induce an ovine PH-RVF model that utilizes left pulmonary artery (LPA) ligation, progressive main pulmonary artery (MPA) banding, and insertion of an RV pressure line for monitoring. The PA cuff and RV pressure tubing are connected to subcutaneous access ports. This model of PH-RVF is a versatile platform to control not only the disease severity, but also the RV's phenotypic response. Subjects undergo progressive PA band adjustments twice per week for approximately 9weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). Subjects can further be exercised on a livestock treadmill while hemodynamic parameters are captured. At the initiation and endpoint of this model, ventricular function and dimensions are assessed using echocardiography. In this model, RV mean and systolic pressure increased to 28±5 and 57±7mmHg at week 1, and further to 44±7 and 93±18mmHg by week 9, respectively. Echocardiography demonstrates characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The rate of PA banding has a significant impact on SvO2 and thus the model can be titrated to elicit varying RV phenotypes. When the PA cuff is tightened rapidly, it can lead to a precipitous decline in SvO2, leading to RV decompensation, whereas a slower, more paced strategy leads to an adaptive RV stress-load response that maintains physiologic SvO2. A faster rate of PA banding will also lead to more severe liver fibrosis. The addition of controlled exercise provides a useful platform for assessing the effects of physical exertion in a PH-RVF model. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and evaluating mechanical circulatory support systems.

Ca2+ handling remodelling in a porcine model of right ventricular dysfunction

Increased afterload is the primary pathophysiologic mechanism for right ventricular failure (RVF), which is the most critical determinant of survival in patients with pulmonary hypertension (PH). The cellular and molecular processes involved in the RV remodeling, including Ca2+ handling as an instrumental actor in the heart, remain incompletely understood. We gain insights into the cellular Ca2+ signaling in a large animal model of RV failure, the chronic thromboembolic pulmonary hypertension (CTEPH) piglet model, to develop specific therapy targeting RVF. CTEPH piglet model consisted of a primary left pulmonary artery ligation followed by weekly embolization in the right lower lobe leading to PH and progressive RV hypertrophy and failure. After six weeks, the piglet model replicated all the features of human CTEPH: increased pulmonary vascular resistance, mean pulmonary arterial pressure (PAP), and RV wall thickness associated with RV dysfunction as assessed by the reduced tricuspid annular plane systolic excursion (TAPSE). RV histological analyses revealed hypertrophied cardiomyocytes and aberrant fibrosis. At the cell level, RV myocytes from CTEPH piglets presented reduced L-type Ca2+ currents and CACNA1C mRNA, lower and slower [Ca2+]i transients, decreased sarcoplasmic reticulum Ca2+ content, and cell shortening to myocytes from sham piglets. This was related to reduce Sarco/Endoplasmic Reticulum Ca2+-ATPase isoform 2a (SERCA2a) protein expression. We found that store-operated Ca2+ entry is increased in hypertrophied RV myocytes, which is correlated with a de novo expression of the SR Ca2+ sensor STIM1L (long stromal interacting molecule 1). Finally, we found that Ca2+ sparks are larger and longer in RV myocytes from CTEPH piglets, whereas their frequencies and amplitude were unchanged compared to myocytes from sham. This was associated with profound disorganization of T tubules and ryanodine receptors (RyR) clustering without changes in RyR phosphorylation status. Our data highlight a cellular Ca2+ cycling remodeling that participates to the pathogenesis of RVF in CTEPH piglets.

Microbiome and metabolome dysbiosis of the gut-lung axis in pulmonary hypertension

Previous studies have suggested that dysbiosis of the gut microbiota is associated with the development of pulmonary hypertension (PH). In this study, we established a left pulmonary artery ligation (LPAL)-induced PH rat model due to high flow and hemodynamic stress and investigated the association between gut microbiota composition and host metabolome signatures (in both gut and lung tissues) by using multiomics and correlation analysis. The results showed that LPAL successfully induced PH, characterized by increased right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodelling. Moreover, gut pathological abnormalities were observed in association with dramatic alterations in the gut microbiome and metabolome as well as the lung metabolome. The increased bacterial genus Sporobacter and decreased genera Eubacterium, Eubacteriaceae, Deltaproteobacteria and Desulfovibrio featured the altered gut microbiome in LPAL-PH versus SHAM rats. Moreover, imbalanced abundance of protective metabolites (e.g., butyrate, propionate) and pathogenic metabolites (e.g., proinflammatory mediators) were seen in the gut metabolome of LPAL-PH versus SHAM rats. In addition, the altered gut microbiome strongly correlated with the altered metabolome patterns in both the gut and lung of LPAL-PH rats. In conclusion, this study revealed significant gut dysbiosis in LPAL-PH rats, characterized by altered gut microbiota composition, in association with specific changes in gut and lung metabolome profiles. These findings enriched our understanding of the unique signature of the gut microbiome and the close association of the “gut-lung axis” in LPAL-PH induced by long-term high flow, leading to novel therapeutic, diagnostic or management paradigms for this subtype of PH.

Upregulation of mechanosensitive channel Piezo1 involved in high shear stress-induced pulmonary hypertension

IntroductionPiezo1 is an important mechanosensitive channel implicated in vascular remodeling. However, the role of Piezo1 in different types of vascular cells during the development of pulmonary hypertension (PH) induced by high shear stress is largely unknown. Materials and methodsWe used a rat PH model established by left pulmonary artery ligation (LPAL, for 2–5 weeks), which mimics the high flow and hemodynamic stress, to study Piezo1 contribution to pulmonary vascular remodeling. ResultsRight ventricular systolic pressure (RVSP), a surrogate measure for pulmonary arterial systolic pressure, and right ventricular wall thickness, a measure for right ventricular hypertrophy, were significantly increased in LPAL rats compared with Sham-control (SHAM) rats. Rats in LPAL-5w groups developed remarkable pulmonary vascular remodeling, while phenylephrine-induced contraction and acetylcholine-induced relaxation were both significantly inhibited in these rats. Upregulation of Piezo1, in association with increase in cytosolic Ca2+ concentration ([Ca2+]cyt), was observed in pulmonary arterial smooth muscle cells (PASMCs) from LPAL-2w and LPAL-5w rats in comparison to the SHAM controls. Piezo1 upregulation in PASMCs from LPAL rats was directly related to Yes-associated protein (YAP)/ TEA domain transcription factor 4 (TEAD4). Piezo1 expression was also upregulated in the whole-lung tissue of LPAL rats. The endothelial upregulation of Piezo1 was related to transcriptional regulation by RELA (p65) and lung inflammation. ConclusionThe upregulation of Piezo1 in both PASMCs and ECs coordinates with each other via different cell signaling pathways to cause pulmonary vascular remodeling in LPAL-PH rats, providing novel insights into the cell-type specific pathogenic roles of Piezo1 in shear stress-associated experimental PH.

Shock lung is not “wet” but characterized as necroptotic inflammation in a mouse model of hypotension

ObjectivesHypotension episodes before or after donor brain death are assumed to trigger hypoxia-reoxygenation, causing diffuse alveolar-capillary damage via necrosis. However, alveolar-capillary membranes have direct access to oxygen in alveoli. We hypothesized hypotension-induced lung injury is not diffuse alveolar-capillary damage but interstitial inflammation resulting from nonhypoxic lung ischemia and systemic responses to hypoxic extrapulmonary ischemia. MethodsThe 4-hour hypotension model was established by subjecting C57BL/6J mice to 4-hour hypotension at 15 ± 5 mm Hg of mean artery pressure and resuscitated with whole shed blood and norepinephrine. Nonhypoxic lung ischemia model was established by 4-hour left pulmonary artery ligation. At 24 hours postprocedure, an arterial blood gas analysis and a gastroduodenal occult blood test were conducted. Lung samples were assessed for histology, cytokine transcripts, regulated cell death, and alveolar-capillary permeability. ResultsThe 4-hour hypotension model had an intraoperative mortality rate of 17.7% (41/231) and a stress-ulcer bleeding rate of 15.3% (29/190). No signs of alveolar flooding were observed in both models. Four-hour hypotension without stress ulcer showed normal oxygenation and permeability but increased interstitial infiltration, transcription of Tnf and Il1b, phosphorylation of MLKL and RIPK3, and cleaved caspase 3 compared with 4-hour pulmonary artery ligation and naïve control. Animals that developed stress ulcer presented with worse pulmonary infiltration, intracellular edema, and oxygenation but just slightly increased permeability. Immunoblotting showed significant upregulations of protein expression and phosphorylation of MLKL and RIPK3, cleaved Caspase-3, but not its prototype in 4-hour hypotension with stress ulcer. ConclusionsHypotensive lung injury is essentially a nonhypoxic ischemia–reperfusion injury enhanced by systemic responses. It is predominated by necroptosis-induced inflammation rather than necrosis-induced diffuse alveolar-capillary damage.

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 µg/kg/min to 7.5 µg/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV's phenotypic response. Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 ± 5 and 57 ± 7 mmHg at week 1, respectively, to 44 ± 7 and 93 ± 18 mmHg (mean ± standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 < 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Addition of 5% CO2 to Inspiratory Gas Prevents Lung Injury in an Experimental Model of Pulmonary Artery Ligation.

Rationale: Unilateral ligation of the pulmonary artery may induce lung injury through multiple mechanisms, which might be dampened by inhaled CO2.Objectives: This study aims to characterize bilateral lung injury owing to unilateral ligation of the pulmonary artery in healthy swine undergoing controlled mechanical ventilation and its prevention by 5% CO2 inhalation and to investigate relevant pathophysiological mechanisms.Methods: Sixteen healthy pigs were allocated to surgical ligation of the left pulmonary artery (ligation group), seven to surgical ligation of the left pulmonary artery and inhalation of 5% CO2 (ligation + FiCO2 5%), and six to no intervention (no ligation). Then, all animals received mechanical ventilation with Vt 10 ml/kg, positive end-expiratory pressure 5 cm H2O, respiratory rate 25 breaths/min, and FiO2 50% (±FiCO2 5%) for 48 hours or until development of severe lung injury.Measurements and Main Results: Histological, physiological, and quantitative computed tomography scan data were compared between groups to characterize lung injury. Electrical impedance tomography and immunohistochemistry analysis were performed in a subset of animals to explore mechanisms of injury. Animals from the ligation group developed bilateral lung injury as assessed by significantly higher histological score, larger increase in lung weight, poorer oxygenation, and worse respiratory mechanics compared with the ligation + FiCO2 5% group. In the ligation group, the right lung received a larger fraction of Vt and inflammation was more represented, whereas CO2 dampened both processes.Conclusions: Mechanical ventilation induces bilateral lung injury within 48 hours in healthy pigs undergoing left pulmonary artery ligation. Inhalation of 5% CO2 prevents injury, likely through decreased stress to the right lung and antiinflammatory effects.

Left Pulmonary Artery Ligation and Chronic Pulmonary Artery Banding Model for Inducing Right Ventricular-Pulmonary Hypertension in Sheep.

Pulmonary hypertension (PH) is a progressive disease that leads to cardiopulmonary dysfunction and right heart failure from pressure and volume overloading of the right ventricle (RV). Mechanical cardiopulmonary support has theoretical promise as a bridge to organ transplant or destination therapy for these patients. Solving the challenges of mechanical cardiopulmonary support for PH and RV failure requires its testing in a physiologically relevant animal model. Previous PH models in large animals have used pulmonary bead embolization, which elicits unpredictable inflammatory responses and has a high mortality rate. We describe a step-by-step guide for inducing pulmonary hypertension and right ventricular hypertrophy (PH-RVH) in sheep by left pulmonary artery (LPA) ligation combined with progressive main pulmonary artery (MPA) banding. This approach provides a controlled method to regulate RV afterload as tolerated by the animal to achieve PH-RVH, while reducing acute mortality. This animal model can facilitate evaluation of mechanical support devices for PH and RV failure.

Ligation of left pulmonary artery instead of patent ductus arteriosus.

Ductus arteriosus is an essential component of fetal circulation. Due to occurring changes in the cardiopulmonary system physiology after birth, ductus arteriosus closes. Patent ductus arteriosus can be closed by medical or invasive (percutaneous or surgical) treatment methods. Percutaneous or surgical closure of patent ductus arteriosus can be performed for the cases that medical closure failed. Surgical treatment is often preferred method for closure of patent ductus arteriosus in the neonatal period. The most common surgical complications are pneumothorax, recurrent laryngeal nerve injury, bleeding, and recanalisation. A very rare surgical complication is left pulmonary artery ligation that has been presented in a few cases in the literature. Echocardiography control should be performed in the early post-operative period, especially in patients with clinical suspicion. If reoperation is required, it should never be delayed. We report a newborn patient whose left pulmonary artery ligated accidentally during patent ductus arteriosus closure surgery and surgical correction of this complication at the early post-operative period.

Ovine Models of Congenital Heart Disease and the Consequences of Hemodynamic Alterations for Pulmonary Artery Remodeling.

The natural history of pulmonary vascular disease associated with congenital heart disease (CHD) depends on associated hemodynamics. Patients exposed to increased pulmonary blood flow (PBF) and pulmonary arterial pressure (PAP) develop pulmonary vascular disease more commonly than patients exposed to increased PBF alone. To investigate the effects of these differing mechanical forces on physiologic and molecular responses, we developed two models of CHD using fetal surgical techniques: 1) left pulmonary artery (LPA) ligation primarily resulting in increased PBF and 2) aortopulmonary shunt placement resulting in increased PBF and PAP. Hemodynamic, histologic, and molecular studies were performed on control, LPA, and shunt lambs as well as pulmonary artery endothelial cells (PAECs) derived from each. Physiologically, LPA, and to a greater extent shunt, lambs demonstrated an exaggerated increase in PAP in response to vasoconstricting stimuli compared with controls. These physiologic findings correlated with a pathologic increase in medial thickening in pulmonary arteries in shunt lambs but not in control or LPA lambs. Furthermore, in the setting of acutely increased afterload, the right ventricle of control and LPA but not shunt lambs demonstrates ventricular-vascular uncoupling and adverse ventricular-ventricular interactions. RNA sequencing revealed excellent separation between groups via both principal components analysis and unsupervised hierarchical clustering. In addition, we found hyperproliferation of PAECs from LPA lambs, and to a greater extent shunt lambs, with associated increased angiogenesis and decreased apoptosis in PAECs derived from shunt lambs. A further understanding of mechanical force-specific drivers of pulmonary artery pathology will enable development of precision therapeutics for pulmonary hypertension associated with CHD.

Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia

ABSTRACTPurpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Materials and Methods: Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague–Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A2A (A2AAR) and A2B adenosine receptor (A2BAR) mRNA and protein were quantified. Results: Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A2BAR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A2AAR mRNA and protein concentrations remained unchanged following ischemia. A2BAR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Conclusion: Increased A2BAR and adenosine following unilateral lung ischemia as well as more BOOP in A2BAR mutant rats implicate a protective role for A2BAR signaling in countering ischemic lung injury.

Effector T Cells and Ischemia-Induced Systemic Angiogenesis in the Lung.

Lymphocytes have been shown to modulate angiogenesis. Our previous work showed that T regulatory (Treg) cell depletion prevented angiogenesis. In the present study, we sought to examine T-cell populations during lung angiogenesis and subsequent angiostasis. In a mouse model of ischemia-induced systemic angiogenesis in the lung, we examined the time course (0-35 d) of neovascularization and T-cell phenotypes within the lung after left pulmonary artery ligation (LPAL). T cells increased and reached a maximum by 10 days after LPAL and then progressively decreased, suggestive of a modulatory role during the early phase of new vessel growth. Because others have shown IFN-γ to be angiostatic in tumor models, we focused on this effector T-cell cytokine to control the magnitude of angiogenesis. Results showed that IFN-γ protein is secreted at low levels after LPAL and that mice required Treg depletion to see the full effect of effector T cells. Using Foxp3(DTR) and diphtheria toxin to deplete T regulatory cells, increased numbers of effector T cells (CD8(+)) and/or increased capacity to secrete the prominent angiostatic cytokine IFN-γ (CD4(+)) were seen. In vitro culture of mouse systemic and pulmonary microvascular endothelial cells with IFN-γ showed increased endothelial cell apoptosis. CD8(-/-) mice and IFN-γR(-/-) mice showed enhanced angiogenesis compared with wild-type mice, confirming that, in this model, IFN-γ limits the extent of systemic neovascularization in the lung.

Upregulation of canonical transient receptor potential channel in the pulmonary arterial smooth muscle of a chronic thromboembolic pulmonary hypertension rat model.

Chronic ligation of the left main pulmonary artery (PA) results in pulmonary vascular remodeling and sustained vasoconstriction. This method has been used to generate a postobstructive pulmonary vasculopathy model to mimic severe chronic thromboembolic pulmonary hypertension (CTEPH). The aim of this study was to examine the cellular and molecular mechanisms underlying CTEPH and to provide evidence for potential treatments. The CTEPH rat model was induced by surgical left PA ligation (LPAL). Right ventricular systolic pressure (RVSP), lung histochemistry and plasma D-dimer measurements were carried out to evaluate the model. A fluorescence microscope was used to measure the basal intracellular Ca(2+) concentration ([Ca(2+)]i) and store-operated Ca(2+) entry (SOCE) in rat distal pulmonary arterial smooth muscle cells through a Fura-2 fluorescence-based method. The expression of the canonical transient receptor potential channel 1 (TRPC1) and TRPC6 was determined by western blotting and real-time quantitative PCR in isolated distal pulmonary arteries (PA). At the time points of 2 and 5 weeks postsurgery, the RVSP showed significant increases in the LPAL groups in comparison with the respective control groups. LPAL also led to right ventricular hypertrophy (RVH), distal pulmonary arterial remodeling in unobstructed territories and persistently higher plasma D-dimer levels. Increases of the basal [Ca(2+)]i and SOCE in LPAL were associated with a clear upregulation of TRPC1 and TRPC6 expression in the distal PA. Our study demonstrated that LPAL successfully reproduced the vascular tone changes that mimic CTEPH pathogenesis. In this model, the increased RVSP and RVH are likely related to enhanced SOCE and upregulated TRPC1 and TRPC6 expression levels in the distal PA.

Pulmonary microvascular lesions regress in reperfused chronic thromboembolic pulmonary hypertension

Pulmonary microvascular disease (PMD) develops in both occluded and non-occluded territories in patients with chronic thromboembolic pulmonary hypertension (CTEPH) and may cause persistent pulmonary hypertension after pulmonary endarterectomy. Endothelin-1 (ET-1) and interleukin-6 (IL-6) are potential PMD severity biomarkers, but it remains unknown whether they are related to occluded or non-occluded territories. We assessed PMD and ET-1/IL-6 gene expression profiles in occluded and non-occluded territories with and without chronic lung reperfusion in an animal CTEPH model. Chronic PH was induced in 10 piglets by left pulmonary artery (PA) ligation followed by weekly embolization of right lower lobe arteries with enbucrilate tissue adhesive for 5 weeks. At Week 6, 5 of 10 animals underwent left PA reperfusion. At Week 12, animals with and without reperfusion were compared with sham animals (n = 5). Hemodynamics, lung morphometry and ET-1/IL-6 gene expression profiles were assessed in the left lung (LL, occluded territories) and right upper lobe (RUL, non-occluded territories). At Week 12, mean PA pressure remained elevated without reperfusion (29.0 ± 2.8 vs 27.0 ± 1.1 mm Hg, p = 0.502), but decreased after reperfusion (30.0 ± 1.5 vs 20.5 ± 1.7 mm Hg, p = 0.013). Distal media thickness in the LL and RUL PAs and systemic vasculature to the LL were significantly lower in the reperfused and sham groups compared with the non-reperfused group. PMD progression was related to ET-1 and IL-6 gene expression in the RUL and to the ET-A/ET-B gene expression ratio in the LL. PMD regressed in occluded and non-occluded territories after lung reperfusion. Changes in ET-1 and IL-6 gene expression were associated with PMD in non-occluded territories.

Characterization of early neovascular response to acute lung ischemia using simultaneous 19F/1H MR molecular imaging

Angiogenesis is an important constituent of many inflammatory pulmonary diseases, which has been unappreciated until recently. Early neovascular expansion in the lungs in preclinical models and patients is very difficult to assess noninvasively, particularly quantitatively. The present study demonstrated that (19)F/(1)H MR molecular imaging with αvβ3-targeted perfluorocarbon nanoparticles can be used to directly measure neovascularity in a rat left pulmonary artery ligation (LPAL) model, which was employed to create pulmonary ischemia and induce angiogenesis. In rats 3days after LPAL, simultaneous (19)F/(1)H MR imaging at 3T revealed a marked (19)F signal in animals 2h following αvβ3-targeted perfluorocarbon nanoparticles [(19)F signal (normalized to background)=0.80±0.2] that was greater (p=0.007) than the non-targeted (0.30±0.04) and the sham-operated (0.07±0.09) control groups. Almost no (19)F signal was found in control right lung with any treatment. Competitive blockade of the integrin-targeted particles greatly decreased the (19)F signal (p=0.002) and was equivalent to the non-targeted control group. Fluorescent and light microscopy illustrated heavy decorating of vessel walls in and around large bronchi and large pulmonary vessels. Focal segmental regions of neovessel expansion were also noted in the lung periphery. Our results demonstrate that (19)F/(1)H MR molecular imaging with αvβ3-targeted perfluorocarbon nanoparticles provides a means to assess the extent of systemic neovascularization in the lung.

Chemokine Localization in Bronchial Angiogenesis

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0–24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.