Immunostaining For Von Willebrand Factor Research Articles

Immunohistochemical analysis of von Willebrand factor expression in myocardial tissues from autopsies of patients with ischemic heart disease.

von Willebrand factor (VWF) plays a crucial role in hemostasis and thrombosis. VWF is involved in platelet attachment to the subendothelium, serving as a carrier protein for coagulation factor VIII. In this study, myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry to determine VWF expression. We examined 28 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained from the autopsies of patients who were diagnosed with ischemic heart disease within 48h postmortem. Most myocardial cells were negative for VWF, although some cells showed nonspecific positivity. Elevated VWF expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, completely fibrotic myocardial foci did not show upregulated VWF expression. Positivity in fibrin deposition and hemorrhagic sites was observed. The same VWF expression characteristics as those observed in the human samples were observed in the mouse model. VWF immunostaining as an endothelial marker may be a useful supplementation to conventional staining techniques that are currently used in the diagnosis of ischemic heart disease in terms of examining the timing of myocardial remodeling in detail and highlighting the remodeling process.

The Role of Von Willebrand Factor in the Pathogenesis of Pulmonary Vascular Thrombosis in COVID-19.

The increased plasma levels of von Willebrand factor (VWF) in patients with COVID-19 was reported in many studies, and its correlation with disease severity and mortality suggest its important role in the pathogenesis of thrombosis in COVID-19. We performed histological and immunohistochemical studies of the lungs of 29 patients who died from COVID-19. We found a significant increase in the intensity of immunohistochemical reaction for VWF in the pulmonary vascular endothelium when the disease duration was more than 10 days. In the patients who had thrombotic complications, the VWF immunostaining in the pulmonary vascular endothelium was significantly more intense than in nonsurvivors without thrombotic complications. Duration of disease and thrombotic complications were found to be independent predictors of increased VWF immunostaining in the endothelium of pulmonary vessels. We also revealed that bacterial pneumonia was associated with increased VWF staining intensity in pulmonary arterial, arteriolar, and venular endothelium, while lung ventilation was an independent predictor of increased VWF immunostaining in arterial endothelium. The results of the study demonstrated an important role of endothelial VWF in the pathogenesis of thrombus formation in COVID-19.

Rare Type 1-like and Type 2-like Calrecticulin Mutants Induce Similar Myeloproliferative Neoplasms Than Type 1 and Type 2 Mutants in Mice

IntroductionThe acquisition of somatic mutations in the hematopoietic stem cell (HSC) compartment leading to the constitutive activation of the JAK-STAT signaling pathway drives the development of myeloproliferative neoplasms (MPNs). After the gain-of-function mutation JAK2 V617F, the second most frequent driver mutations inducing 30% of essential thrombocythemia and mylofibrosis (MF) are located in the calreticulin (CALR) gene. The two most frequent CALR mutations are a deletion of 52 base pairs (del52) L367fs*46 also called type 1 and an insertion of 5 base pairs (ins5) K385fs*47 or type 2. They represent together more than 80% of all CALR mutations. In retroviral mouse models CALR mutants induce a thrombocytosis, but to a lesser extent in presence of ins5 compared to del52. Moreover, del52 mice evolved more frequently into MF than ins5 mice, mimicking what is observed in patients. Many other rare mutations of CALR exist and each represents less than 2% of CALR mutations in patients. They are structurally classified as type 1- and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha helix structure but it is not known whether they are equally functionally related to CALRdel52 and CALRins5, respectively.Material and MethodsWe modeled two type 1-like mutations, del34 (E369fs*50) and del46 (L367fs*48) and of one type 2-like mutation del19 (K374fs*50) in cell lines and in mice by bone marrow transplantation after retroviral transduction with CALR mutation constructs.ResultIn Ba/F3 cell lines, all CALR mutants specifically activated cytokine-independent cell growth in presence of the thrombopoietin (TPO) receptor (MPL) with close efficiency, and induced a constitutive activation of JAK2 and STAT5/3/1 in a similar way.Lethally irradiated mice that were engrafted with bone marrow transduced with the wild-type (CALR wt) or the different CALR mutations developed pure thrombocytosis but to a much lesser extent in presence of type 2 and type 2-like CALR mutations. The thrombocytosis was associated with megakaryocyte hyperplasia in the bone marrow and in the spleen seen by Von Willebrand factor immunostaining and flow cytometry. In contrast to type 2-like mice, type 1-like mice developed a MF after 10 months of engraftment associated with splenomegaly, a marked osteogenesis in bone marrow and a thickening of reticulin fiber network seen in histology.Like observed for type 1 mutant, type 1-like CALR mutations induced an expansion at early stage of hematopoiesis compare to type 2 and type 2-like mutations with a significant increased frequency in immature SLAM, LSK and MkP progenitors. Altogether, type 1-like CALR mutants harbor a stronger phenotype than the type 2-like recapitulating thrombocytosis and progression to MF.Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble type 1 and type 2, respectively. The mechanisms responsible for these differences remain to be determined and could be related to a MPL independent process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/-. These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

Repeatable and objective method for evaluating angiogenesis using real-time RT-PCR of endoglin expression in canine tumours.

Anti-angiogenic therapy is a cancer treatment strategy targeting new blood vessel formation. Microvessel density (MVD) is a histopathological method for evaluating angiogenesis and endoglin is used as an activated endothelial marker in human medicine. The assessment of the treatment effect using MVD is difficult because it is a non-repeatable method. To develop a repeatable method for evaluating angiogenesis, we investigated correlations among MVD, mRNA transcription levels of endothelial markers and angiogenesis factors, and confirmed the agreement of mRNA transcription levels between tissue samples and small samples obtained by fine needle aspiration (FNA). The various types of spontaneous tumours were collected from 51 dogs. MVD was assessed by immunostaining for von Willebrand factor (vWF). mRNA transcription levels of vWF, endoglin, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analysed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR). There were significant correlations between MVD and mRNA transcription levels of vWF, endoglin and VEGFR2. VEGFR2 was more strongly correlated with endoglin (P <.01, Rs = 0.649) than vWF (P <.01, Rs = 0.512), indicating that angiogenesis can be evaluated more accurately by the measurement of mRNA transcription levels of endoglin. The mRNA transcription levels in tissue and FNA samples were strongly correlated, suggesting that evaluating angiogenesis using FNA samples is possible. In conclusion, we developed a repeatable and objective method for angiogenesis evaluation using mRNA transcription levels of endothelial markers by FNA sampling.

Abstract WP268: Von Willebrand Factor Expression in Various Subtypes of Acute Ischemic Stroke

Introduction: Von Willebrand factor (VWF) is a key component of acute ischemic stroke (AIS) thrombi. The aim of our prospective study was to investigate the immunohistochemical expression of VWF in clots and to evaluate whether VWF is associated with certain subtypes of AIS. Methods: VWF immunostaining was performed on 79 thrombi collected as part of the multi-center Stroke Thromboembolism Registry of Imaging and Pathology (STRIP) registry. The cases were classified according to TOAST criteria. The VWF expression was quantified using Orbit Image Analysis (www.Orbit.bio) machine learning software. IBM SPSS statistics 25 was used to assess the relationship between the VWF levels and different etiology subtypes. Results: A cardioembolic stroke was defined in 39 cases (49.4%) whereas an atherosclerotic origin was identified in 13 patients (16.5%). Other causes accounted for 12 cases (15.1%). Unknown etiology was reported in 15 cases (19%). The mean VWF content in the clots was 12.8%. According to the Mann-Whitney U-test, the level of VWF was significantly higher in the cases with unknown etiology compared to cardioembolic origin (p=0.044). We found also that patients with unknown etiology of stroke had higher VWF expression as compared to the other two subtypes, although this difference was not statistically significant. Conclusions: Among the patients with ischemic stroke included in this study, the VWF expression was significantly increased in those with unknown etiology compared to the group with cardioembolic stroke. Our finding provides new insights into clot composition in cryptogenic stroke and may influence the treatment and secondary prevention in these cases.

Effects of Fibroblast Growth Factor-2 Combined With a Collagen/Gelatin Sponge for Adipogenesis in the Mouse Subcutis.

A collagen/gelatin sponge (CGS) is a new scaffold that promotes wound healing by slowly releasing fibroblast growth factor (FGF)-2. FGF-2 induces mitogenesis, angiogenesis, and adipogenesis. In this study, the adipogenesis-inducing effects of CGS combined with FGF-2 in the subcutis of mice were evaluated. Collagens/gelatin sponges (10 × 5 mm) were impregnated with 50 μL of FGF-2 solution (10 or 100 μg/mL). A CGS (Gunze Corp, Osaka, Japan) combined with FGF-2 was implanted subcutaneously into the thoracic region of mice. At 1, 2, 3, and 4 weeks, samples were collected for hematoxylin and eosin staining, von Willebrand factor immunostaining, and perilipin immunostaining to examine adipose tissue localization and angiogenesis. A CGS with only saline solution was prepared as a control. Adipocytes in the collagen fibers appeared at 3 weeks, and a zonal fat layer was noted under the panniculus carnosus at 4 weeks in the FGF-2-combined CGS groups. The fat layer was significantly thicker in the FGF-2 (100 μg/mL) group than in the FGF-2 (10 μg/mL) group. In the control group, no fat pad was newly formed. The number of newly formed vessels in the FGF (10 μg/mL) and (100 μg/mL) groups was significantly greater in the FGF-2 group than in the control group. This study presents a promising method to enhance adipogenic effects in the murine subcutis using CGS combined with FGF-2, representing a potential technique for soft tissue reconstruction.

FGF-2 combined with bilayer artificial dermis composed of collagen matrix prompts generation of fat pad in subcutis of mice

Fibroblast growth factor (FGF)-2 induces mitogenesis, angiogenesis and adipogenesis. In this study, the adipogenesis-inducing effects of FGF-2 combined with bilayer artificial dermis in mice were evaluated. FGF-2-impregnated bilayer artificial dermis composed of collagen matrix, PELNAC (Gunze Corp., Osaka, Japan) was implanted subcutaneously into the thoracic region of mice. At 1, 2, 3, and 4 weeks, samples were collected for H&E staining, von Willebrand factor immunostaining, and perilipin immunostaining to examine adipose tissue localization and angiogenesis. The collagen matrix-implanted group without the addition of FGF-2 was prepared as a control. At 2 weeks after the implantation of FGF-2 combined with dermal substitutes, adipocytes appeared in the collagen fibers. At 3–4 weeks, a fat pad was generated with neovascularization. The thickness of the fat pad had significantly increased at 2, 3, and 4 weeks. The remaining collagen was decreased by absorption over time. In the control group, no fat pad was newly formed. This study has identified a promising method to enhance adipogenic effects in the murine subcutis, representing a potential technique for soft tissue reconstruction.

Placental Growth Factor Contributes to Liver Inflammation, Angiogenesis, Fibrosis in Mice by Promoting Hepatic Macrophage Recruitment and Activation

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, mediates wound healing and inflammatory responses, exerting an effect on liver fibrosis and angiogenesis; however, the precise mechanism remains unclear. The aims of this study are to identify the role of PlGF in liver inflammation and fibrosis induced by bile duct ligation (BDL) in mice and to reveal the underlying molecular mechanism. PlGF small interfering RNA (siRNA) or non-targeting control siRNA was injected by tail vein starting 2 days after BDL. Liver inflammation, fibrosis, angiogenesis, macrophage infiltration, and hepatic stellate cells (HSCs) activation were examined. Our results showed that PlGF was highly expressed in fibrotic livers and mainly distributed in activated HSCs and macrophages. Furthermore, PlGF silencing strongly reduced the severity of liver inflammation and fibrosis, and inhibited the activation of HSCs. Remarkably, PlGF silencing also attenuated BDL-induced hepatic angiogenesis, as evidenced by attenuated liver endothelial cell markers CD31 and von Willebrand factor immunostaining and genes or protein expression. Interestingly, these pathological ameliorations by PlGF silencing were due to a marked reduction in the numbers of intrahepatic F4/80+, CD68+, and Ly6C+ cell populations, which were reflected by a lower expression of these macrophage marker molecules in fibrotic livers. In addition, knockdown of PlGF by siRNA inhibited macrophages activation and substantially suppressed the expression of pro-inflammatory cytokines and chemokines in fibrotic livers. Mechanistically, evaluation of cultured RAW 264.7 cells revealed that VEGF receptor 1 (VEGFR1) mainly involved in mediating the role of PlGF in macrophages recruitment and activation, since using VEGFR1 neutralizing antibody blocking PlGF/VEGFR1 signaling axis significantly inhibited macrophages migration and inflammatory responses. Together, these findings indicate that PlGF plays an important role in liver inflammation, angiogenesis, and fibrosis by promoting hepatic macrophage recruitment and activation, and suggest that blockage of PlGF could be a promising novel therapy for chronic fibrotic liver diseases.

Abstract 493: Syndesomes: An Syndecan-4 Based Therapeutic for Effective Revascularization in Peripheral Ischemia in Diabetes

Previous research has attempted to use growth factor proteins and genes to revascularize ischemic tissues but none of the methods have found complete success in clinical trials. We have demonstrated the loss of co-receptors like syndecan-4 due to long-term diseased state and enhanced neovascularization of ischemic tissue due to co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 in normal healthy rats. In this study, we used syndesomes to enhance revascularization in the ischemic hind limb of a growth factor resistant mouse model with diabetes, obesity and hyperlipidemia. We used ob/ob mice fed with high fat diet for 10 weeks. We induced ischemia through femoral artery ligation and implanted alginate beads containing treatments into the surgical site. The recovery of perfusion in the mice was tracked using laser speckle imaging. After 14 days, the ischemic limbs of the mice treated with FGF-2 with syndesomes had significantly higher perfusion compared to the FGF-2 alone group (A, B). Morphometric analysis on sections from the calf and thigh muscle of the mice demonstrated increased ischemic changes in the ischemic limbs of the FGF-2 alone group. Immunostaining for von Willebrand factor revealed increased neovascularization corresponding with the increase perfusion measured in the FGF-2 with syndesomes group (C, D). Immunostaining for pro-inflammatory M1 macrophage marker CD86 showed similar levels in both treatment groups (E, F). However, immunostaining for pro-wound healing M2 macrophage marker revealed significantly higher levels in the syndesome with FGF-2 treatment group (G, H). Taken together, these preliminary studies support that co-delivery of syndecan-4 with FGF-2 significantly enhances revascularization in the ischemic hind limb in a clinically relevant diseased mouse model. Our treatment restores the signaling pathway components that are lost due to diseased state causing tissues to become resistant to growth factor therapies.

Early systemic microvascular damage in pigs with atherogenic diabetes mellitus coincides with renal angiopoietin dysbalance.

BackgroundDiabetes mellitus (DM) is associated with a range of microvascular complications including diabetic nephropathy (DN). Microvascular abnormalities in the kidneys are common histopathologic findings in DN, which represent one manifestation of ongoing systemic microvascular damage. Recently, sidestream dark-field (SDF) imaging has emerged as a noninvasive tool that enables one to visualize the microcirculation. In this study, we investigated whether changes in the systemic microvasculature induced by DM and an atherogenic diet correlated spatiotemporally with renal damage.MethodsAtherosclerotic lesion development was triggered in streptozotocin-induced DM pigs (140 mg/kg body weight) by administering an atherogenic diet for approximately 11 months. Fifteen months following induction of DM, microvascular morphology was visualized in control pigs (n = 7), non-diabetic pigs fed an atherogenic diet (ATH, n = 5), and DM pigs fed an atherogenic diet (DM+ATH, n = 5) using SDF imaging of oral mucosal tissue. Subsequently, kidneys were harvested from anethesized pigs and the expression levels of well-established markers for microvascular integrity, such as Angiopoietin-1 (Angpt1) and Angiopoietin-2 (Angpt2) were determined immunohistochemically, while endothelial cell (EC) abundance was determined by immunostaining for von Willebrand factor (vWF).ResultsOur study revealed an increase in the capillary tortuosity index in DM+ATH pigs (2.31±0.17) as compared to the control groups (Controls 0.89±0.08 and ATH 1.55±0.11; p<0.05). Kidney biopsies showed marked glomerular lesions consisting of mesangial expansion and podocyte lesions. Furthermore, we observed a disturbed Angpt2/ Angpt1balance in the cortex of the kidney, as evidenced by increased expression of Angpt2 in DM+ATH pigs as compared to Control pigs (p<0.05).ConclusionIn the setting of DM, atherogenesis leads to the augmentation of mucosal capillary tortuosity, indicative of systemic microvascular damage. Concomitantly, a dysbalance in renal angiopoietins was correlated with the development of diabetic nephropathy. As such, our studies strongly suggest that defects in the systemic microvasculature mirror the accumulation of microvascular damage in the kidney.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), d-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32–80 years) or duration in eye bank corneal preservation medium prior to use (3–10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries

Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium.

Prevention of lipopolysaccharide-induced peritoneal damage by eplerenone in rats undergoing peritoneal dialysis

Bacterial peritonitis in patients undergoing peritoneal dialysis (PD) is a major cause of therapy interruption due to peritoneal insufficiency. Here we studied the effect of a selective mineralocorticoid receptor (MR) blocker, eplerenone, on the prevention of peritoneal damage. Male Sprague-Dawley rats were treated with a daily infusion of human use PD solution (100 mL/kg i.p., PD group, n = 5), or with PD solution and intermittent intraperitoneal injections of lipopolysaccharide (LPS group, n = 5) or with LPS and eplerenone (100 mg/ kg/d, po, Ep group, n = 5) for 4 weeks. Peritoneal samples were subjected to assessment following the peritoneal equilibration test (PET). Histological observations revealed that LPS treatment resulted in significant peritoneal thickening associated with increased ED-1-positive cell infiltration and the number of transforming growth factor (TGF)-β1-positive cells, and that eplerenone reduced these changes. LPS administration also evoked significant upregulation of monocyte chemotactic protein-1 and TGF-β1, which were inhibited by eplerenone. PET revealed that ultrafiltration and transperitoneal osmotic diffusion were significantly impaired by LPS and restored by eplerenone. Increased value of the mass transfer area coefficients for creatinine values was also recovered by Ep (0.10 ± 0.01 in the PD, 0.14 ± 0.02 in the LPS and 0.08 ± 0.0 in the Ep groups). Immunostaining for von Willebrand factor showed a significant increase by LPS and its restoration by Ep. Ep effectively diminished LPS-induced peritoneal insufficiency. A selective blockade of MR might prevent peritoneal insufficiency associated with bacterial peritonitis.

S114 Lymphatic vessel distribution in fibrotic lung diseases

Relatively little is known on lymphatic vessel remodelling in lung fibrosis, and whether differences in lymphatic distribution underlie the worse survival seen in idiopathic pulmonary fibrosis, compared to the other...

Angiotensin II type 2 receptor signaling significantly attenuates growth of murine pancreatic carcinoma grafts in syngeneic mice

BackgroundPancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. To evaluate the effect of angiotensin II (Ang II) type 2 receptor (AT2) expression in the host's body on the growth of pancreatic carcinoma, we have investigated the growth of mouse pancreatic ductal carcinoma grafts in syngeneic wild type and AT2 receptor-deficient (AT2-KO) mice.MethodsThe role of AT2 receptor-signaling in stromal cells on the growth of murine pancreatic carcinoma cells (PAN02) was studied using various in vitro and in vivo assays. In vivo cell proliferation, apoptosis, and vasculature in tumors were monitored by Ki-67 immunostaining, TUNEL assay, and von Willebrand factor immunostaining, respectively. In the co-culture study, cell proliferation was measured by MTT cell viability assay. All the data were analyzed using t-test and data were treated as significant when p < 0.05.ResultsOur results show that the growth of subcutaneously transplanted syngeneic xenografts of PAN02 cells, mouse pancreatic ductal carcinoma cells derived from the C57/BL6 strain, was significantly faster in AT2-KO mice compared to control wild type mice. Immunohistochemical analysis of tumor tissue revealed significantly more Ki-67 positive cells in xenografts grown in AT2-KO mice than in wild type mice. The index of apoptosis is slightly higher in wild type mice than in AT2-KO mice as evaluated by TUNEL assay. Tumor vasculature number was significantly higher in AT2-KO mice than in wild type mice. In vitro co-culture studies revealed that the growth of PAN02 cells was significantly decreased when grown with AT2 receptor gene transfected wild type and AT2-KO mouse-derived fibroblasts. Faster tumor growth in AT2-KO mice may be associated with higher VEGF production in stromal cells.ConclusionsThese results suggest that Ang II regulates the growth of pancreatic carcinoma cells through modulating functions of host stromal cells; Moreover, Ang II AT2 receptor signaling is a negative regulator in the growth of pancreatic carcinoma cells. These findings indicate that the AT2 receptor in stromal fibroblasts is a potentially important target for chemotherapy for pancreatic cancer.

Assessment of myocardial blood perfusion improved by CD151 in a pig myocardial infarction model

To appraise the efficacy of CD151-induced myocardial therapeutic angiogenesis in a pig myocardial infarction model. CD151 and anti-CD151 were constructed into the recombinant adeno-associated virus (rAAV) vector. All 26 pigs were subjected to coronary artery ligation or no surgery. Eight weeks after coronary artery ligation, the expression of CD151 was measured by Western blot and immunostaining. Capillary density was evaluated using immunostaining for von Willebrand factor (vWF). 13N-labeled NH3 positron emission computed tomography ([13N]NH3PET) was measured to assess regional myocardial perfusion and the defect area. CD151 gene delivery could increase the expression of CD151 at protein level. Over-expression of CD151 increased the density of total capillaries in the ischemic myocardium, significantly improved the blood perfusion and reduced the defect area percentage. This study demonstrated that the rAAV-mediated CD151 gene delivery promoted efficient neovascularization and increased the blood perfusion after myocardial infarction in pigs.

Role of VEGF in small bowel adaptation after resection: the adaptive response is angiogenesis dependent

Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 +/- 8.816 vs. 310 +/- 19.35, P = 0.01) and crypt depth (100.021 +/- 4.025 vs. 120.541 +/- 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 +/- 0.29 vs. 6.1 +/- 0.29 vs. 5.96 +/- 0.20) and villus-vessel area ratio (0.713 +/- 0.01 vs. 0.73 +/- 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response.

Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.

Vein Graft Harvesting Induces Inflammation and Impairs Vessel Reactivity

Saphenous veins are often used for coronary artery bypass grafting (CABG), but loss of patency is a problem. The surgical procedure may contribute to graft injury. Our aim was to study the impact of surgical handling of saphenous veins on graft inflammation and vascular function. Biopsy samples of saphenous veins were taken from 9 patients undergoing elective CABG at the start of vein harvesting (open technique) and after the last proximal anastomosis was sutured. Messenger RNA was extracted and amplified with semiquantitative reverse transcription polymerase chain reaction. Gene expression of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta), leukocyte adhesion molecules (E-selectin, intercellular adhesion molecule-1), and vasoactive substances (endothelin-1, inducible and endothelial nitric oxide synthase) was investigated. Translocation of nuclear factor-kappaB (NFkappaB) was evaluated with electrophoretic mobility shift assay. Immunostaining for von Willebrand factor was performed to evaluate loss of endothelium, and in vitro vein reactivity to phenylephrine and endothelin-1 was studied. Gene expression of cytokines and leukocyte adhesion molecules increased after graft harvesting and storage, whereas vasoactive substances did not change. Nuclear translocation of NFkappaB occurred after surgical handling, concurrent with partial loss of endothelium and impaired contractile function. Standard surgical handling of vein grafts induces NFkappaB-driven inflammation in the vessel wall and impairs vascular function. This may potentially contribute to both early and late graft occlusion.