Plexiform Lesions Research Articles

Pulmonary vasculopathy in explanted lungs from patients with interstitial lung disease undergoing lung transplantation

BackgroundPulmonary hypertension (PH) causes increased morbidity and mortality in patients with interstitial lung diseases (ILD). Classification schemes, while well-characterised for the vasculopathy of idiopathic PH, have been applied, unchallenged, to...

The Uterine Plexiform Lesions Revisited.

Uterine lesions with plexiform morphology are uncommon lesions with debated histogenesis. Despite being an incidental and usually benign finding (plexiform tumorlet), some cases can pose diagnostic problems. Their paucity in the recent literature adds to these difficulties and often causes ambiguities. The objective of this study is to systematically review published cases to highlight the historical aspects of their recognition, reappraising their morphology, histogenesis, and differential diagnosis. English literature is reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, and 32 reports are analyzed. Most cases are reported in the fourth to sixth decades. In most cases (66.7%), plexiform lesions are incidental findings while 33.3% of cases have been the chief pathology. Size varies from 0.5 to 195 mm. Plexiform foci were solitary in 78.2% cases and multiple in 21.8%. In 67.8% of cases, the lesions are reported as myometrial, while 32.2% are arising from endometrial stroma. Immunohistochemistry shows smooth muscle and no sex cord marker expression. They are usually benign lesions, but worrisome features include plexiform morphology in disseminated peritoneal leiomyomatosis, intravenous leiomyomatosis, and diffuse uterine leiomyomatosis. Plexiform lesions represent a diverse pathology varying from epithelioid leiomyomas to epithelioid smooth muscle metaplasia of endometrial type of stroma.

Endomyocardial fibrosis.

Endomyocardial fibrosis (EMF) is a neglected cardiovascular disease of poverty which carries a poor prognosis with no specific treatment affecting mainly children and young adults. Here, we report our 10-year experience in the therapeutic management and surgical treatment for EMF. From February 2009 to 2019 March, 55 patients diagnosed with EMF from our cardiology unit underwent surgical repair at our department's pediatric surgical division. There were 35 male, and 20 female patients whose ages varied from 1 year 2 months to 12 years mean age 5.7 (±3.2). We designed the study aimed at assessing the cardio-structural abnormalities and coronary vascular changes faced with EMF patients using echocardiography, and coronary angiography with a detailed and thorough surgical examination of each case. Of the 55 operated patients, 1 had mild lesions, 26 had moderate lesions, and 28 had severe heart disease. All but one patient was in NYHA functional class III or IV at the time of surgery. All but one female patient with mild ventricular lesions and no valvular involvement had severe atrioventricular valve regurgitation with valves considered suitable for both replacements; 45 patients mean age 6.0 (±3.1) and repair nine patients mean age 3.8 (±2.9). The mean endocardial thickness was 3,000 (±1519) µm. The echocardiographic changes corresponded well to the findings on surgery and histopathology. The coronary changes seen included a spectrum of fibrin deposition, medial sclerosis and degeneration, and the formation of plexiform lesions. Surgically evaluating the resected cardiac tissue might help improve disease management.

O-GlcNAc Transferase Regulates Angiogenesis in Idiopathic Pulmonary Arterial Hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is considered a vasculopathy characterized by elevated pulmonary vascular resistance due to vasoconstriction and/or lung remodeling such as plexiform lesions, the hallmark of the PAH, as well as cell proliferation and vascular and angiogenic dysfunction. The serine/threonine hydroxyl-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) has been shown to drive pulmonary arterial smooth muscle cell (PASMC) proliferation in IPAH. OGT is a cellular nutrient sensor that is essential in maintaining proper cell function through the regulation of cell signaling, proliferation, and metabolism. The aim of this study was to determine the role of OGT and O-GlcNAc in vascular and angiogenic dysfunction in IPAH. Primary isolated human control and IPAH patient PASMCs and pulmonary arterial endothelial cells (PAECs) were grown in the presence or absence of OGT inhibitors and subjected to biochemical assessments in monolayer cultures and tube formation assays, in vitro vascular sprouting 3D spheroid co-culture models, and de novo vascularization models in NODSCID mice. We showed that knockdown of OGT resulted in reduced vascular endothelial growth factor (VEGF) expression in IPAH primary isolated vascular cells. In addition, specificity protein 1 (SP1), a known stimulator of VEGF expression, was shown to have higher O-GlcNAc levels in IPAH compared to control at physiological (5 mM) and high (25 mM) glucose concentrations, and knockdown resulted in decreased VEGF protein levels. Furthermore, human IPAH PAECs demonstrated a significantly higher degree of capillary tube-like structures and increased length compared to control PAECs. Addition of an OGT inhibitor, OSMI-1, significantly reduced the number of tube-like structures and tube length similar to control levels. Assessment of vascular sprouting from an in vitro 3D spheroid co-culture model using IPAH and control PAEC/PASMCs and an in vivo vascularization model using control and PAEC-embedded collagen implants demonstrated higher vascularization in IPAH compared to control. Blocking OGT activity in these experiments, however, altered the vascular sprouting and de novo vascularization in IPAH similar to control levels when compared to controls. Our findings in this report are the first to describe a role for the OGT/O-GlcNAc axis in modulating VEGF expression and vascularization in IPAH. These findings provide greater insight into the potential role that altered glucose uptake and metabolism may have on the angiogenic process and the development of plexiform lesions. Therefore, we believe that the OGT/O-GlcNAc axis may be a potential therapeutic target for treating the angiogenic dysregulation that is present in IPAH.

Aneurysm-type plexiform lesions form in supernumerary arteries in pulmonary arterial hypertension: potential therapeutic implications.

Histological observations in human pulmonary arterial hypertension (PAH) suggest a link between plexiform lesions and pulmonary supernumerary arteries. Pulmonary microvascular endothelial cells are characterized as hyperproliferative and progenitor-like. This study investigates the hypothesis that aneurysm-type plexiform lesions form in pulmonary supernumerary arteries because of their anatomical properties and endothelial characteristics similar to pulmonary microvascular endothelial cells. To induce PAH, rats were injected with Sugen5416, and exposed to hypoxia (10% O2) for 3 days (early stage) or 3 wk (mid-stage), or 3 wk of hypoxia with an additional 10 wk of normoxia (late-stage PAH). We examined morphology of pulmonary vasculature and vascular remodeling in lung serial sections from PAH and normal rats. Aneurysm-type plexiform lesions formed in small side branches of pulmonary arteries with morphological characteristics similar to supernumerary arteries. Over the course of PAH development, the number of Ki67-positive cells increased in small pulmonary arteries, including supernumerary arteries, whereas the number stayed consistently low in large pulmonary arteries. The increase in Ki67-positive cells was delayed in supernumerary arteries compared with small pulmonary arteries. In late-stage PAH, ~90% of small unconventional side branches that were likely to be supernumerary arteries were nearly closed. These results support our hypothesis that supernumerary arteries are the predominant site for aneurysm-type plexiform lesions in Sugen5416/hypoxia/normoxia-exposed PAH rats partly because of the combination of their unique anatomical properties and the hyperproliferative potential of endothelial cells. We propose that the delayed and extensive occlusive lesion formation in supernumerary arteries could be a preventive therapeutic target in patients with PAH.

Endovascular cell therapy in a model of arteriovenous malformation: Concept of Bio-Embolization

Arteriovenous malformations (AVM) are complex plexiform vascular lesions. Complete surgical excision is the treatment of choice but it is often unachievable. Embolo-sclerotherapy alone is associated with an important rate of recurrence. Our assumption was that the endovascular administration of mesenchymal stem cells (BM-MSCs) conditioned in a hydrogel in the nidus of an AVM could induce a solid microvascular occlusion by endoluminal tissue growth.

Intratracheal SERCA2a Gene Therapy Interrupts the Formation of Plexiform Lesions and Rescues from Heart Failure in Severe Pulmonary Arterial Hypertension

Introduction Drugs fail to improve long term outcomes in pulmonary hypertension (PAH), especially survival in end stages. Severe PAH presents with deadly high pulmonary vascular resistance [PVR] due to the progression of irreversible plexiform lesions [PLX], resulting in heart failure [HF]. SERCA2a [S2A], a calcium handling protein, has been shown effective in PAH via reprogramming endothelial and vascular smooth muscle cells via improving pulmonary flow and disrupting neointimal proliferation. Preliminary S2A studies have shown efficacy in reducing mean PA pressure [mPAP], however in less severe PAH models without HF or PLX. Here, we used a severe surgical PAH HF model and evaluated S2A gene therapy. Hypothesis S2A gene therapy will reduce PVR and rescue from HF via reducing the degree of PLX. Methods Twenty rats were divided between control or treatment groups, n=10 ea. All rats were induced with severe PAH via left pneumonectomy combined with Sugen (25mg/kg). Grade 4 maximum plexiform lesions develop with mPAP >35 mmHg @ 3 weeks. AAV1.S2A was administered via intratracheal aerosol at 2.5 × 1012 VGC after 3 weeks. MRI was performed at baseline, 3 and 6 weeks terminal with invasive mPAP. Harvest assays performed: QPCR of AAV1.S2A genome copies, S2A expression, and PLX pathology. Results S2A rats survived at 80% compared to control 50%. QPCR detection was very high in 5 S2A animals [1314±858 GC/cell], remaining 3 with average 2.5 fold in pulmonary smooth muscle anatomy 1B. Unlike control, which presented with higher frequency Grade 4 PLX 1C, the majority of S2A specimens had Grade 1-3 with less occurrence of PLX 1D. Conclusion S2A gene therapy is effective in reducing the progression of PLX. Gene therapy may serve as an adjunctive option for severe PAH patients.

LRP1 Deficiency in Vascular SMC Leads to Pulmonary Arterial Hypertension That Is Reversed by PPARγ Activation.

Arterial remodeling-a hallmark of many cardiovascular pathologies including pulmonary arterial hypertension (PAH)-is regulated by TGFβ1 (transforming growth factor-β1)-TGFβ receptors and the antagonistic, vasoprotective BMPR2 (bone morphogenetic protein receptor 2)-PPARγ (peroxisome proliferator-activated receptor-γ) axis. However, it is unclear which factors drive detrimental TGFβ1 pathways in the hypertensive pulmonary vasculature. We hypothesized that LRP1 (low-density lipoprotein receptor-related protein 1) expression is decreased in PAH, leading to enhancement (disinhibition) of TGFβ1 signals and that the PPARγ agonist pioglitazone can restore vascular homeostasis and prevent PAH resulting from LRP1 deletion in vascular smooth muscle cells (SMCs). Targeted deletion of LRP1 in vascular SMC (smLRP1-/-) in mice disinhibited TGFβ1-CTGF (connective tissue growth factor) signaling, leading to spontaneous PAH and distal pulmonary arterial muscularization as assessed by closed-chest cardiac catheterization and anti-αSMA staining. Pioglitazone inhibited the canonical TGFβ1-CTGF axis in human pulmonary artery SMC and smLRP1-/- main pulmonary artery (CTGF and NOX4) and reversed PAH in smLRP1-/- mice. TGFβ1 boosted pSmad3 in PASMC from smLRP1-/- mice versus controls. Pioglitazone-activated PPARγ binds to Smad3 in human pulmonary artery SMC (coimmunoprecipitation), thereby blocking its phosphorylation and overriding LRP1 deficiency. Finally, mRNA and protein expression of LRP1 was decreased in pulmonary plexiform lesions of patients with end-stage idiopathic PAH (laser capture microdissection, qPCR, and immunohistochemistry). Downregulation of LRP1 protein was also demonstrated in explanted PASMC from patients with PAH and accompanied by enhanced TGFβ1-pSmad3-CTGF signaling and increased TGFβ1-induced PASMC proliferation that was prevented by pioglitazone. Here, we identify LRP1 as an integrator of TGFβ1-mediated mechanisms that regulate vascular remodeling in mice and clinical PAH and PPARγ as a therapeutic target that controls canonical TGFβ1 pathways. Hence, pharmacologic PPARγ activation represents a promising new therapy for patients with PAH who lack the vasoprotective LRP1 in vascular SMC.

AGE-RAGE Stress in the Pathophysiology of Pulmonary Hypertension and its Treatment.

Pulmonary hypertension (PH) is a rare and fatal disease characterized by elevation of pulmonary artery pressure ≥ 25 mm Hg. There are five groups of PH: (1) pulmonary artery (PA) hypertension (PAH), (2) PH due to heart diseases, (3) PH associated with lung diseases/hypoxia, (4) PH associated with chronic obstruction of PA, and (5) PH due to unclear and/or multifactorial mechanisms. The pathophysiologic mechanisms of group 1 have been studied in detail; however, those for groups 2 to 5 are not that well known. PH pathology is characterized by smooth muscle cells (SMC) proliferation, muscularization of peripheral PA, accumulation of extracellular matrix (ECM), plexiform lesions, thromboembolism, and recanalization of thrombi. Advanced glycation end products (AGE) and its receptor (RAGE) and soluble RAGE (sRAGE) appear to be involved in the pathogenesis of PH. AGE and its interaction with RAGE induce vascular hypertrophy through proliferation of vascular SMC, accumulation of ECM, and suppression of apoptosis. Reactive oxygen species (ROS) generated by interaction of AGE and RAGE modulates SMC proliferation, attenuate apoptosis, and constricts PA. Increased stiffness in the artery due to vascular hypertrophy, and vasoconstriction due to ROS resulted in PH. The data also suggest that reduction in consumption and formation of AGE, suppression of RAGE expression, blockage of RAGE ligand binding, elevation of sRAGE levels, and antioxidants may be novel therapeutic targets for prevention, regression, and slowing of progression of PH. In conclusion, AGE-RAGE stress may be involved in the pathogenesis of PH and the therapeutic targets should be the AGE-RAGE axis.

Development of anti-angiogenic erlotinib liposomal formulation for pulmonary hypertension: a QbD approach.

Pulmonary arterial hypertension (PAH) is the increase in mean pulmonary arterial pressure (> 25mmHg). The development of the non-reversible plexiform lesions on the arterial walls of the pulmonary arteries has evolved as the reason to increase the pressure. The current treatments are directed towards the vasodilation of the pulmonary arteries via the endothelin, prostacyclin, and NO pathways which provides symptomatic relief. Deeper understanding of the disease leads to the various pathophysiological targets that play an important role in the development of PAH. Out of these, the angiogenetic mechanism of the pulmonary arterial smooth muscle cells has been proved to play an important role in PAH. Targeted therapies by anti-proliferative drugs may lead to the efficient treatment strategies to the root cause of PAH. Erlotinib, a receptor tyrosine kinase inhibitor, which acts on the epidermal growth factor receptor (EGFR), has shown promising results in clinical trials of PAH. The objective of the work has been the development of liposomal formulation of anti-proliferative drug, erlotinib HCl, via Quality by Design (QbD) approach. The liposomal formulation was developed using thin-film hydration technique and characterised for various physicochemical parameters, like particle size, % entrapment efficiency, DSC, FTIR, pXRD, and TEM. In the drug release study, the formulation showed sustained release of erlotinib over 24h in simulated lung fluid pH7.4. This developed formulation was evaluated in zebrafish tail fin regeneration assay for its anti-angiogenetic activity. The liposomal formulation inhibited the tail fin regeneration for 14days indicating anti-angiogenetic activity.

Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension.

Uncontrolled proliferation of endothelial cells is essential to the pathogenesis of pulmonary arterial hypertension (PAH). Both proliferation and cytoskeleton reorganization are associated with upregulation of the intermediate filament protein Nestin. Recently, accumulation of Nestin-expressing cells was found in pulmonary vascular lesions of PAH patients. The goal of this study is to determine if Nestin expression contributes to endothelial proliferation in pulmonary arterial hypertension, using both lung tissues and endothelial cells. Here we found that endothelial cells from complex and plexiform lesions of PAH patients expressed Nestin. These Nestin+ cells further stained positive for the angiogenic factors CXC chemokine ligand 12 and Wnt1. Likewise, in the chronic hypoxia/SU5416 animal model of pulmonary hypertension, Nestin+ endothelial cells were found in occlusive pulmonary vascular lesions. In vitro, both growing rat and human lung endothelial cells expressed Nestin protein. When Nestin was overexpressed in endothelial cells (both rat and human), Nestin overexpression promoted proliferation and expression of CXC chemokine ligand 12. Nestin overexpression further increased angiogenic tube formation in vitro. Conclusions: We found increased Nestin expression from endothelial cells of occlusive lung vascular lesions in severe pulmonary hypertension. Elevated Nestin expression likely contributes to unchecked pulmonary vascular proliferation and angiogenesis, possibly via induction of CXC chemokine ligand 12. Additional studies are required to determine whether targeting Nestin would be beneficial to treat PAH.

Sarcoidosis Associated Pulmonary Hypertension: Correlation of Cascular Morphologic Changes with Hemodynamic Data in Explanted Lungs

Purpose Sarcoidosis associated pulmonary hypertension (PH) is classified as WHO Group V according to the 2018 WHO classification system. Previous case series have demonstrated efficacy of pulmonary vasodilators in treating severe PH in sarcoidosis. We present a series of patients with sarcoidosis associated PH and describe correlation between the pre-transplant hemodynamic data from a right heart catheterization (RHC) and vascular morphologic changes in the native explanted lungs. Methods Patients with sarcoidosis who underwent lung transplantation at Loyola University Medical Center were analyzed from 2014-2018. We collected demographic data, RHC data, NYHA functional class data, and echocardiogram. Pathology was reviewed to assess for presence and severity of fibrosis, presence or absence of granulomas in lung parenchyma and lymph nodes, and vascular morphologic changes using Heath Edwards PH classification. We evaluated PH changes in arterioles, muscular arteries of 1mm and 3mm caliber, and elastic arteries. These findings were correlated to RHC data. Results Seven patients who had a RHC prior to lung transplantation were identified and had a mean pulmonary artery pressure greater than 25 mmHg. All of the patients had well-formed granulomas except two cases that showed ill formed granulomas. 5 of 7 cases had lymph nodes involved and 5 of 7 had fibrosis. Medial hypertrophy of 1 and 3mm arteries was observed in all cases. 3 of 4 patients with severe PH (NYHA Class IV) and RV dysfunction on echocardiogram were on parenteral prostacyclin therapy at the time of transplant and had grade 3 changes in the 1 mm arteries and grade 2 changes in the 3 mm arteries. Only 2 of 7 cases showed arteriolar changes, which correlated with higher pressures. No plexiform lesions were seen in any of the patients. Conclusion In our series of sarcoidosis patients with PH who underwent lung transplant or transplantation, vascular morphologic changes at the level of the 1 mm and arterioles correlated with the presence of severe PH (NYHA Class IV) and RV dysfunction on echocardiogram. These patients lack the plexiform lesions typically seen in idiopathic pulmonary arterial hypertension. Our study may identify a subset of sarcoid patients who have significant vascular remodeling and possibly explains the recently observed favorable response to vasodilator therapy.

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The Search for Disease-Modifying Therapies in Pulmonary Hypertension.

Pulmonary hypertension (PH) and its severe subtype pulmonary arterial hypertension (PAH) encompass a set of multifactorial diseases defined by sustained elevation of pulmonary arterial pressure and pulmonary vascular resistance leading to right ventricular failure and subsequent death. Pulmonary hypertension is characterized by vascular remodeling in association with smooth muscle cell proliferation of the arterioles, medial thickening, and plexiform lesion formation. Despite our recent advances in understanding its pathogenesis and related therapeutic discoveries, PH still remains a progressive disease without a cure. Nevertheless, development of drugs that specifically target molecular pathways involved in disease pathogenesis has led to improvement in life quality and clinical outcomes in patients with PAH. There are presently more than 12 Food and Drug Administration-approved vasodilator drugs in the United States for the treatment of PAH; however, mortality with contemporary therapies remains high. More recently, there have been exuberant efforts to develop new pharmacologic therapies that target the fundamental origins of PH and thus could represent disease-modifying opportunities. This review aims to summarize recent developments on key signaling pathways and molecular targets that drive PH disease progression, with emphasis on new therapeutic options under development.

Human PAH is characterized by a pattern of lipid-related insulin resistance.

Pulmonary arterial hypertension (PAH) is a deadly disease of the small pulmonary vasculature with an increased prevalence of insulin resistance (IR). Insulin regulates both glucose and lipid homeostasis. We sought to quantify glucose- and lipid-related IR in human PAH, testing the hypothesis that lipoprotein indices are more sensitive indices of IR in PAH. Oral glucose tolerance testing in PAH patients and triglyceride-matched (TG-matched) controls and proteomic, metabolomics, and lipoprotein analyses were performed in PAH and controls. Results were validated in an external cohort and in explanted human PAH lungs. PAH patients were similarly glucose intolerant or IR by glucose homeostasis metrics compared with control patients when matched for the metabolic syndrome. Using the insulin-sensitive lipoprotein index, TG/HDL ratio, PAH patients were more commonly IR than controls. Proteomic and metabolomic analysis demonstrated separation between PAH and controls, driven by differences in lipid species. We observed a significant increase in long-chain acylcarnitines, phosphatidylcholines, insulin metabolism-related proteins, and in oxidized LDL receptor 1 (OLR1) in PAH plasma in both a discovery and validation cohort. PAH patients had higher lipoprotein axis-related IR and lipoprotein-based inflammation scores compared with controls. PAH patient lung tissue showed enhanced OLR1 immunostaining within plexiform lesions and oxidized LDL accumulation within macrophages. IR in PAH is characterized by alterations in lipid and lipoprotein homeostasis axes, manifest by elevated TG/HDL ratio, and elevated circulating medium- and long-chain acylcarnitines and lipoproteins. Oxidized LDL and its receptor OLR1 may play a role in a proinflammatory phenotype in PAH. NIH DK096994, HL060906, UL1 RR024975-01, UL1 TR000445-06, DK020593, P01 HL108800-01A1, and UL1 TR002243; American Heart Association 13FTF16070002.

Experimental intravascular hemolysis induces hemodynamic and pathological pulmonary hypertension: association with accelerated purine metabolism.

Pulmonary hypertension (PH) is emerging as a serious complication associated with hemolytic disorders, and plexiform lesions (PXL) have been reported in patients with sickle cell disease (SCD). We hypothesized that repetitive hemolysis per se induces PH and angioproliferative vasculopathy and evaluated a new mechanism for hemolysis-associated PH (HA-PH) that involves the release of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) from erythrocytes. In healthy rats, repetitive administration of hemolyzed autologous blood (HAB) for 10 days produced reversible pulmonary parenchymal injury and vascular remodeling and PH. Moreover, the combination of a single dose of Sugen-5416 (SU, 200 mg/kg) and 10-day HAB treatment resulted in severe and progressive obliterative PH and formation of PXL (Day 26, right ventricular peak systolic pressure (mmHg): 26.1 ± 1.1, 41.5 ± 0.5 and 85.1 ± 5.9 in untreated, HAB treated and SU+HAB treated rats, respectively). In rats, repetitive administration of HAB increased plasma ADA activity and reduced urinary adenosine levels. Similarly, SCD patients had higher plasma ADA and PNP activity and accelerated adenosine, inosine, and guanosine metabolism than healthy controls. Our study provides evidence that hemolysis per se leads to the development of angioproliferative PH. We also report the development of a rat model of HA-PH that closely mimics pulmonary vasculopathy seen in patients with HA-PH. Finally, this study suggests that in hemolytic diseases released ADA and PNP may increase the risk of PH, likely by abolishing the vasoprotective effects of adenosine, inosine and guanosine. Further characterization of this new rat model of hemolysis-induced angioproliferative PH and additional studies of the role of purines metabolism in HA-PH are warranted.

Prevalence of ALK gene alterations among the spectrum of plexiform spitzoid lesions

ALK receptor tyrosine kinase gene (ALK) rearrangements have been described in spitzoid lesions with a plexiform growth pattern. To investigate the prevalence of ALK alterations in a large series of spitzoid lesions. ALK immunohistochemical and fluorescence in situ hybridization analyses of 78 spitzoid plexiform lesions including 41 Spitz nevi, 29 atypical Spitz tumors (ASTs), and 8 spitzoid melanomas. ALK immunohistochemical staining was observed in 14.6% of Spitz nevi (6 of 41) and 13.8% of ASTs (4 of 29); the spitzoid melanomas were ALK negative. Fluorescence in situ hybridization confirmed ALK translocation in 9 cases and amplification in 1 case. In 2 of the translocated cases it was possible to determine the fusion partner gene (ie, tropomyosin 3 gene [TPM3] or dynactin 1 gene [DCTN1]). Of the 4 cases of AST examined, 2 carried the B-Raf proto-oncogene, serine/threonine kinase gene (BRAF) V600E mutation. The 10 patients had a mean age of 18.7years (range, 1-39) and a female predominance (female-to-male ratio, 7:3). Seven lesions arose on the extremities; the 2 lesions occurring in infants were located on the face. The lesions' mean diameter was 6.2mm (range, 3-13), and their mean Breslow thickness was 1.83mm (range, 0.6-3.6). The results of sentinel node biopsy were negative in 2 ASTs. BRAF status was tested in only 4 of 10 samples because of the limited amount of material. ALK alterations characterize a significant subset of spitzoid lesions.

Establishment and evaluation of a new method for determining hemodynamics of pulmonary hypertension rats

Objective: By evaluating the hemodynamic parameters such as cardiac output (CO), right ventricular pressure (RVP), pulmonary artery pressure (PAP) and total pulmonary resistance index (TPRI) in pulmonary hypertension rat model, we established a more comprehensive hemodynamic evaluation system, which objectively evaluated the severity of disease and exercise tolerance in rats with pulmonary hypertension. Methods: SD rats were randomly divided into a control group and a model group with 5 rats in each group. The model group was intraperitoneally injected with SU5416 (20 mg/kg) and placed in an oxygen chamber at a 10% oxygen concentration for 21 days and then placed in a normoxic environment for 14 days. After modeling, rats were anesthetized and mechanically ventilated. The operator cut the skin along the right paraxial line, detached and ligated the intercostal artery, and then cut off the 3 and 4 ribs, exposing the heart and freeing aortic root about 0.2 cm. The flowmeter probe was set in the dissected aortic segment, and real-time recording time, blood flow waveforms, cardiac output were calculated accordingly. Then the needle attached to the baroreceptor was inserted into the right ventricle and the system acquired the right ventricular time-pressure waveform. After the waveform stabilized for about 30 seconds, the end of the cannula was sent to the pulmonary artery trunk through the entrance of the pulmonary artery to record the time-pressure curve of the pulmonary artery. Results: RVSP, PASP, PADP and mPAP in the model group were significantly higher than those of the control group [ RVSP(23.4±5.4) mmHg, 1 mmHg=0.133 kPa vs (56.4±13.0) mmHg, PASP (22.8±4.4) mmHg vs (58.5±14.9) mmHg, PADP (9.7±1.9) mmHg vs (30.3±7.0) mmHg, mPAP (14.1±2.7) mmHg vs (41.9±8.0) mmHg, all P<0.05 ]. Compared with the control group, the cardiac index in the model group was significantly lower [ CI (0.54±0.08) ml·min(-1)·g(-1) vs (0.40±0.09) ml·min(-1)·g(-1,) P=0.02 ]. Furthermore, compared with the control group, pulmonary vascular resistance index was significantly increased in the model group[PVRI (0.27±0.03) mmHg·ml(-1)·min(-1)·kg(-1) vs (0.06±0.01) mmHg·ml(-1)·min(-1)·kg(-1,) P<0.05]. The pathological results also showed that the middle part of pulmonary arterioles in the model group had muscular hypertrophy and muscular pulmonary arterioles, and even plexiform lesions. Conclusion: In this study, we established a new method that simultaneously determined several hemodynamic parameters such as RVSP, PASP, PADP, CO, CI and PVRI, which provided a more comprehensive assessment of hemodynamic changes in pulmonary hypertension rat models.

Expression of angiogenic factors and plexiform lesions in the lungs of broiler and layer chickens: A comparison

Plexiform lesions are characteristic histological changes of pulmonary arteries in human patients with severe pulmonary arterial hypertension (PAH) and are regarded as angiogenic lesions. Meat-type broiler chickens are susceptible to PAH and can develop plexiform lesions spontaneously. Whether the lesion development in broilers is associated with PAH predisposition and lung angiogenic environment remains unclear. Moreover, little is known about the cellular origin of these structures. In this work, plexiform lesions were detected in both layer chickens (a strain known to be resistant to PAH) and broiler chickens aged between 1 and 6 wk with normal pulmonary arterial pressures. Within each of the sampled ages, the lesion density did not differ between strains, with an exception of wk 4 when broiler was higher than layer. In contrast to the trend of age-related decline in layers, lesion densities in broilers demonstrated bi-phasic alterations characterized by a gradual decrease during wk 1 to 3 followed by a sudden increase at wk 4. The mRNA of 6 angiogenic factors in the lung tissue, namely, vascular endothelial growth factor receptor (VEGFR)-2, angiopoietin (Ang)-1, angiopoietin receptor Tie-2, transforming growth factor (TGF)-β1, hepatocyte growth factor (HGF), and interleukin (IL)-8, were differentially expressed between strains. However, none of them was found to be significantly correlated with the lesion density by strain and age-adjusted partial correlation analysis. An in vivo experiment revealed impaired differentiation of endothelial progenitor cells (EPC) into endothelial cells during the producing of plexiform lesions, as evidenced by increased expression of endothelial CD133, a maker of EPC, but reduced expression of CD31, a marker of mature endothelial cells, in the parent vessels of plexiform lesions compared to normal vessels. Collectively, it appears unlikely that the predisposition to PAH or intrapulmonary angiogenic environment contributes to the lesion development in broilers when compared with layers. It is suggested that the lesion development is associated with increased pulmonary arterial pressure, and that local EPC dysfunction may play a role in the process.

Abstract 277: Lung Gene Transfer With Sarcoplasmic Reticulum Calcium ATPase Prevent Disease Progression in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by vascular remodeling leading to high pulmonary arterial pressure (PAP) and right ventricular (RV) heart failure. Gene therapy is a promising approach to treat PAH. The most used monocrotaline (MCT) rat model of PAH does not mimic the pathophysiology of the PAH in humans. A refined rat model, the pneumonectomy plus MCT (PNT-MCT), displays all of the features of PAH and most importantly plexiform lesions whereby therapeutics aim to reverse. In this study, we investigated whether intratracheal delivery of sarcoplasmic reticulum calcium ATPase (SERCA2a) gene reverse the severity of PAH in the PNT- MCT model. Left pneumonectomy was performed in rats. One week later, the animals received the MCT injection. At 3 weeks, the severity of PAH disease was confirmed. Thereafter, PNT-MCT rats received either intratracheal delivery of gene construct with adeno-associated virus/SERCA2a (AAV1/SERCA2a) or saline. Hemodynamic parameters were determined by magnetic resonance imaging (MRI) and RV catheterization. RV hypertrophy, heart and lung fibrosis were assessed. Molecular biology assays for gene expression and immunostaining were used to quantify SERCA2a and disease markers. Four weeks after gene delivery, RV function was improved in AAV1.SERCA2a treated-animals with an increase of stroke volume and ejection fraction compared to saline group (275 ± 22 μl vs. 192 ± 22 μl and 56 ± 3 μl vs. 44 ± 3 μl, p&lt;0.05), while RV end systolic volume was decreased (199 ± 16 μl vs. 283 ± 24 μl, p&lt;0.05). Hemodynamic parameters including mean pulmonary pressure were improved in AAV1.SERCA2a group (26 ± 3 mmHg vs. 61 ± 6 mmHg and 21 ± 3 mmHg vs. 41 ± 3 mmHg respectively, p&lt;0.01) compared to control. RV hypertrophy was reduced in AAV1.SERCA2a treated animals (0.37g ± 0.03 vs. 0.63g ± 0.02, p&lt;0.0001). All serotypes of collagen demonstrated decreased expression after AAV1/SERCA2a administration. Histologically, the animals after gene therapy showed a significant regression of plexiform lesions from grade 4 to grade 1-2. In conclusion, intratracheal administration of AAV1/SERCA2a gene can reverse the severe PAH phenotype and may be considered as a potential treatment.