The dating of thrombus organization in cases of pulmonary embolism: an autopsy study.

BACKGROUND: Pulmonary embolism (PE) is associated to high mortality rate worldwide. However, the diagnosis of PE often results inaccurate. Many cases of PE are incorrectly diagnosed or missed and they are often associated to sudden unexpected death (SUD). In forensic practice, it is important to establish the time of thrombus formation in order to determine the precise moment of death. The autopsy remains the gold standard method for the identification of death cause allowing the determination of discrepancies between clinical and autopsy diagnoses. The aim of our study was to verify the morphological and histological criteria of fatal cases of PE and evaluate the dating of thrombus formation considering 5 ranges of time. METHODS: Pulmonary vessels sections were collected from January 2010 to December 2017. Sections of thrombus sampling were stained with hematoxylin and eosin. The content of infiltrated cells, fibroblasts and collagen fibers were scored using a semi-quantitative three-point scale of range values. RESULTS: The 30 autopsies included 19 males (63.3%) and 11 females (36.7%) with an average age of 64.5 ± 12.3 years. The time intervals were as follows: early (≤1 h), recent (> 1 h to 24 h), recent-medium (> 24 h to 48 h), medium (> 48 h to 72 h) and old (> 72 h). In the first hour, we histologically observed the presence of platelet aggregation by immunofluorescence method for factor VIII and fibrinogen. The presence of lymphocytes has been identified from recent thrombus (> 1 h to 24 h) and the fibroblast cells were peripherally located in vascular tissue between 48 and 72 h, whereas they resulted central and copious after 72 h. CONCLUSIONS: After a macroscopic observation and a good sampling traditional histology, it is important to identify the time of thrombus formation. We identified histologically a range of time in the physiopathology of the thrombus (early, recent, recent-medium, medium, old), allowing to determine the dating of thrombus formation and the exact time of death. CLINICAL TRIAL NUMBER: NCT03887819. TRIAL REGISTRATION: The trial registry is Cliniclatrials.gov, with the unique identifying number NCT03887819. The date of registration was 03/23/2019 and it was "Retrospectively registered".

Collagen Matrix Remodeling in Stented Pulmonary Arteries after Transapical Heart Valve Replacement.

The use of valved stents for minimally invasive replacement of semilunar heart valves is expected to change the extracellular matrix and mechanical function of the native artery and may thus impair long-term functionality of the implant. Here we investigate the impact of the stent on matrix remodeling of the pulmonary artery in a sheep model, focusing on matrix composition and collagen (re)orientation of the host tissue. Ovine native pulmonary arteries were harvested 8 (n = 2), 16 (n = 4) and 24 (n = 2) weeks after transapical implantation of self-expandable stented heart valves. Second harmonic generation (SHG) microscopy was used to assess the collagen (re)orientation of fresh tissue samples. The collagen and elastin content was quantified using biochemical assays. SHG microscopy revealed regional differences in collagen organization in all explants. In the adventitial layer of the arterial wall far distal to the stent (considered as the control tissue), we observed wavy collagen fibers oriented in the circumferential direction. These circumferential fibers were more straightened in the adventitial layer located behind the stent. On the luminal side of the wall behind the stent, collagen fibers were aligned along the stent struts and randomly oriented between the struts. Immediately distal to the stent, however, fibers on both the luminal and the adventitial side of the wall were oriented in the axial direction, demonstrating the stent impact on the collagen structure of surrounding arterial tissues. Collagen orientation patterns did not change with implantation time, and biochemical analyses showed no changes in the trend of collagen and elastin content with implantation time or location of the vascular wall. We hypothesize that the collagen fibers on the adventitial side of the arterial wall and behind the stent straighten in response to the arterial stretch caused by oversizing of the stent. However, the collagen organization on the luminal side suggests that stent-induced remodeling is dominated by contact guidance.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Pulmonary artery pseudoaneurysm caused by a rare vascular tumor: epithelioid hemangioendothelioma.

We present a case of a 58-year-old female with a rare vascular tumor of intermediate malignancy. The initial manifestation was a pseudoaneurysm caused by the rupture of the right pulmonary artery after tumor invasion. The diagnosis of epithelioid hemangioendothelioma was confirmed by the morphologic and immunocytochemical features after surgery. The patient recovered smoothly and there has been no evidence of local recurrence or metastasis during the 2 years of follow-up.

A novel method for detection of apoptosis.

There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

[Association between pulmonary vascular remodeling and expression of hypoxia-inducible factor-1α, endothelin-1 and inducible nitric oxide synthase in pulmonary vessels in neonatal rats with hypoxic pulmonary hypertension].

OBJECTIVE: To investigate the association between pulmonary vascular remodeling and expression of hypoxia-inducible factor-1α (HIF-1α), endothelin-1 (ET-1) and inducible nitric oxide synthase (iNOS) in pulmonary vessels in neonatal rats with hypoxic pulmonary hypertension (HPH). METHODS: A neonatal rat model of HPH was established as an HPH group, and normal neonatal rats were enrolled as a control group. The mean pulmonary arterial pressure (mPAP) was measured. The percentage of medial thickness to outer diameter of the small pulmonary arteries (MT%) and the percentage of medial cross-section area to total cross-section area of the pulmonary small arteries (MA%) were measured as the indicators for pulmonary vascular remodeling. The immunohistochemical reaction intensities for HIF-1α, ET-1 and iNOS and their mRNA expression in lung tissues of neonatal rats were measured. Correlation analysis was performed to determine the relationship between pulmonary vascular remodeling and mRNA expression of HIF-1α, ET-1 and iNOS. RESULTS: The mPAP of the HPH group kept increasing on days 3, 5, 7, 10, 14, and 21 of hypoxia, with a significant difference compared with the control group (P<0.05). The HPH group had significantly higher MT% and MA% than the control group from day 7 of hypoxia (P<0.05). HIF-1α protein expression increased significantly on days 3, 5, 7 and 10 days of hypoxia, and HIF-1α mRNA expression increased significantly on days 3, 5 and 7 days of hypoxia in the HPH group compared with the control group (P<0.05). ET-1 protein expression increased significantly on days 3, 5 and 7 days of hypoxia and ET-1 mRNA expression increased significantly on day 3 of hypoxia in the HPH group compared with the control group (P<0.05). Both iNOS protein and mRNA expression were significantly higher on days 3, 5 and 7 days of hypoxia than the control group (P<0.05). Both MT% and MA% were positively correlated with HIF-1α mRNA expression (r=0.835 and 0.850 respectively; P<0.05). CONCLUSIONS: Pulmonary vascular remodeling is developed on day 7 of hypoxia in neonatal rats. HIF-1α, ET-1 and iNOS are all involved in the occurrence and development of HPH in neonatal rats.

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity.

The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.

Case report of fatal complication in prostatic cryotherapy. First reported death due to argon gas emboli.

We present the first reported fatality from argon gas emboli during prostate cryosurgery. The decedent underwent cryotherapy for prostate carcinoma using cryoablation probes which were cooled with argon and nitrous oxide and warmed with helium. Minutes into the procedure he experienced sudden cardiovascular collapse and could not be resuscitated. Postmortem examination was performed at the request of family and healthcare providers. Collection of tissues and blood samples had to be conducted carefully to capture suspected noble gases,argon, and helium. Specimens were submitted to Saint Louis University Forensic Toxicology Laboratory for toxicological examination and for evaluation of the composition of the gas retrieved from the vascular system.Gas chromatography mass spectrometric analyses confirmed argon in blood, brain, liver, and gas retrieved from the aorta. These samples had significant argon compared with room air also sent for comparison. The manner of death was accident. To date, there have been no intraoperative surgical fatalities reported from prostatic cryotherapy. We report such an unfortunate death to raise awareness in the medical community. We also describe how to collect and handle blood and tissue samples to submit for toxicological analysis in cases of volatile gas emboli.

Homicide due to intravenous metallic mercury injection followed by sodium cyanide injection.

We report a case of homicide due to intravenous mercury injection followed by meperidine and sodium cyanide injection. A 35-year-old woman was found dead in bed at home by her husband. Reportedly, she had been sick for more than 5 months. Initial death investigation revealed no evidence of foul play. Her death was believed to be natural. Therefore, her body was buried without an autopsy. Two months after death, her family requested an autopsy because they suspected her physician husband killed her. Her body was exhumed, and an autopsy was performed. Postmortem examination revealed numerous metallic mercury globules in the pulmonary arteries. Toxicological analysis revealed a high concentration of mercury in the tissue samples of the lungs, liver, heart, and kidney. In addition, cyanide and meperidine were also found in the heart and liver. The detailed case history and postmortem examination findings are described.

Activation of endothelin-1 receptor signaling pathways is associated with neointima formation, neoangiogenesis and irreversible pulmonary artery hypertension in patients with congenital heart disease.

BACKGROUND: It is unclear why some patients, who undergo complete repair or palliative surgery for congenital heart disease (CHD), still develop irreversible pulmonary artery hypertension (PAH). There is no consensus to preoperationally assess the reversible and irreversible pulmonary vasculopathy seen in PAH. METHODS AND RESULTS: The peri-operative pulmonary hemodynamic data of 16 CHD patients (reversible PAH, n = 6; irreversible PAH, n = 10) were analyzed. The lung biopsies were also performed during surgery for defining histopathological characteristics as well as immunohistochemical expression of endothelin-1 (ET-1), endothelin-1 receptors (ETR), and its downstream signaling markers in the small pulmonary arteries and arterioles. Neointimal formation and neoangiogenesis was characterized by increased intimal layer immunoreactivity for α-SMA, Factor VIII, CD34, and VEGF. Neointimal formation was found in 90% of patients and neoangiogenesis was found in 80% of patients with irreversible PAH. Neither was present in the reversible PAH group and the control group. Expression of ET-1 and ETR in the neointimal layer of the pulmonary arterioles was upregulated in irreversible PAH, and immunoreactivity of phospho-Akt, phospho-ERK1/2, and phospho-mTOR was also increased in irreversible PAH. CONCLUSIONS: Increased expression of ET-1, ETR, and activation of signaling pathways were observed in the pulmonary arteries and arterioles of irreversible PAH patients associated with CHD. Activation of these pathways might in turn lead to neointimal formation and neoangiogenesis and thus might contribute to irreversible pulmonary vascular abnormalities.

A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells.

Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes.

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO2-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO2-EtOH-H2O fluids (average molar composition, ΧCO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO2-limonene fluids (ΧCO2 0.88) and neat supercritical CO2 (scCO2) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO2-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO2-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO2-limonene fluids facilitate subsequent enzymatic removal of DNA.

Maladaptive remodeling of pulmonary artery root autografts after Ross procedure: A proteomic study.

OBJECTIVE: Pulmonary autograft root dilatation is the major long-term complication after Ross procedure and the leading cause for reoperation. However, the mechanisms underlying dilatation remain to be elucidated. This study analyzed the proteomic changes seen in the dilated pulmonary autograft compared with normal pulmonary artery and aorta tissues. METHODS: Pulmonary autograft surgical samples were taken from 9 consecutive patients (mean age 37 ± 14; 15-51 years) with mean diameters of 5.2 ± 0.5 cm (4.6-5.8 cm) reoperated 8 to 16 years after Ross procedure. Control pulmonary artery and aorta samples were from 7 age- and sex-matched cardiac donors. Tunicae mediae from all samples were processed for proteomic analysis via 2-dimensional electrophoresis, matrix-assisted-laser-desorption-ionization-time of flight/mass spectrometry, and bioinformatics. The thus-identified putatively relevant proteins were validated via Western immunoblotting. RESULTS: Pulmonary autograft proteome features differed markedly from control pulmonary arteries, since proteins related to focal adhesions (eg, paxillin), cytoskeleton (eg, vimentin), and metalloprotease-regulating proteoglycans (eg, testican-2) were significantly up-regulated, whereas significant decreases occurred in microfibril-associated glycoprotein1, which controls elastic fiber buildup. Profound changes also occurred in cell-signaling proteins, ie, increases in soluble Jagged-1 fragment and ectodysplasin-2 receptor, and decreases in Notch-1 intracellular domain fragment. Moreover, pulmonary autograft expression levels of Paxillin, Vimentin, Jagged-1 fragment, and Notch1 intracellular domain fragment also differed from those of control aorta. CONCLUSIONS: This study provides the first description of the specific proteomic features of dilated pulmonary autograft tunica media, which separate them sharply not only from those of control pulmonary artery and aorta but also of aortic aneurysms. These findings suggest that dilated pulmonary autografts undergo a unique maladaptive remodeling process deserving further investigation.

Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry.

Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 min. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.

Spectral fingerprinting of decellularized heart valve scaffolds.

Decellularized heart valves hold promise for their use as bioscaffolds in cardiovascular surgery. Quality assessment of heart valves after decellularization processing and/or storage is time consuming and destructive. Fourier transform infrared spectroscopy (FTIR) allows rapid non-invasive assessment of biomolecular structures in tissues. In this study, IR-spectra taken from different layers of the pulmonary artery trunk and leaflet tissues of decellularized porcine heart valves were compared with those of pure collagen and elastin, the main protein components in these tissues. In addition, spectral changes associated with aging and oxidative damage were investigated. Infrared absorbance spectra of the arteria intima and media layer were found to be very similar, whereas distinct differences were observed when compared with spectra of the externa layer. In the latter, the shape of the CH-stretching vibration region (3050-2800 cm-1) resembled that of pure collagen. Also, pronounced νCOOH and amide-II bands and a relatively high content of α-helical structures in the externa layer indicated the presence of collagen in this layer. The externa layer of the artery appeared to be sensitive to collagenase treatment, whereas the media and intima layer were particularly affected by elastase and not by collagenase treatment. Protein conformational changes after treatment with collagenase were observed in all three layers. Collagenase treatment completely degraded the leaflet tissue sections. Spectra were also collected from scaffolds after 2 and 12 weeks storage at 37 °C, and after induced oxidative damage. Spectral changes related to aging and oxidative damage were particularly evident in the CH-stretching region, whereas the shape of the amide-I band, reflecting the overall protein secondary structure, remained unaltered.

An autopsy case of pulmonary artery intimal sarcoma: detailed observation of tumor and its related lesions in pulmonary arteries.

We report an autopsy-proven case of a 33-year-old man who died of intimal sarcoma of the pulmonary artery. A large mass (5×4 cm) occluded the main and bilateral pulmonary arteries. Tumor cell morphology was consistent with that of undifferentiated pleomorphic sarcoma. Comprehensive histological observation of 18 pulmonary arteries from proximal to distal revealed continuous extension of the tumor from the main to the subsegmental arteries along the intima, forming an arteriosclerosis-like intimal thickening. Distal small arteries were also affected by eccentric intimal thickening or recanalization. Lung parenchyma was not involved, although there were two wedge-shaped small pulmonary infarctions caused by tumorous obstruction of the associated arteries. Histological results indicated that the intimal sarcoma in the pulmonary artery, which appeared occlusive with growth limited to the proximal artery, had in fact already spread more peripherally than expected. Both the proximal lesions and the distal small arteries were affected by peripheral tumor emboli or by pulmonary hypertension induced by the proximal tumor. However, as seen in this case, most of the occlusive tumor was located locally and intraluminally, in the proximal artery, and removing the proximal tumor by pulmonary endarterectomy was considered effective for symptomatic improvement.

Hypoxia Promotes Vascular Smooth Muscle Cell Proliferation through microRNA-Mediated Suppression of Cyclin-Dependent Kinase Inhibitors.

Regulation of vascular smooth muscle cell (VSMC) proliferation is essential to maintain vascular homeostasis. Hypoxia induces abnormal proliferation of VSMCs and causes vascular proliferative disorders, such as pulmonary hypertension and atherosclerosis. As several cyclin/cyclin-dependent kinase (CDK) complexes and CDK inhibitors (CKIs) control cell proliferation, in this study, we investigated CKIs involved in the hypoxia-induced proliferation process of human primary pulmonary artery smooth muscle cells to understand the underlying molecular mechanism. We demonstrated that p15, p16, and p21 are downregulated in pulmonary artery smooth muscle cells when exposed to hypoxia. In addition, we identified novel hypoxia-induced microRNAs (hypoxamiRs) including miR-497, miR-1268a, and miR-665 that are upregulated under hypoxia and post-transcriptionally regulate p15, p16, and p21 genes, respectively, by directly targeting their 3'UTRs. These miRNAs promoted the proliferation of VSMCs, and their inhibition decreased VSMC proliferation even in hypoxic conditions. Overall, this study revealed that miRNA-mediated regulatory mechanism of CKIs is essential for hypoxia-induced proliferation of VSMCs. These findings provide insights for a better understanding of the pathogenesis of vascular proliferative disorders.

Regulation of the micromechanical properties of pulmonary endothelium by S1P and thrombin: role of cortactin.

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.

Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum.

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.

Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine. FXR may play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. In this study, we report that FXR is also expressed in rat pulmonary artery endothelial cells (EC), a "nonclassical" bile acid target tissue. FXR is functional in EC, as demonstrated by induction of its target genes such as small heterodimer partner (SHP) after treatment with chenodeoxycholic acid, a FXR agonist. Interestingly, activation of FXR in EC led to downregulation of endothelin (ET)-1 expression. Reporter assays showed that activation of FXR inhibited transcriptional activation of the human ET-1 gene promoter and also repressed the activity of a heterologous promoter driven by activator protein (AP)-1 response elements. Electrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR reduced the binding activity of AP-1 transcriptional factors, suggesting that FXR may suppress ET-1 expression via negatively interfering with AP-1 signaling. These studies suggest that FXR may play a role in endothelial homeostasis and may serve as a novel molecular target for manipulating ET-1 expression in vascular EC.