Endothelial Cell-specific Antibodies Research Articles

Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice

BackgroundThe lymphatic vascular pattern in the head of mice has rarely been studied, due to problems of sectioning and immunostaining of complex bony structures. Therefore, the association of head lymphoid tissues with the lymphatics has remained unknown although the mouse is the most often used species in immunology.ResultsHere, we studied the association of nasal and nasolacrimal duct lymphatics with lymphoid aggregates in 14-day-old and 2-month-old mice. We performed paraffin sectioning of whole, decalcified heads, and immunostaining with the lymphatic endothelial cell-specific antibodies Lyve-1 and Podoplanin. Most parts of the nasal mucous membrane do not contain any lymphatics. Only the region of the inferior turbinates contains lymphatic networks, which are connected to those of the palatine. Nose-associated lymphoid tissue (NALT) is restricted to the basal parts of the nose, which contain lymphatics. NALT is continued occipitally and can be found at both sides along the sphenoidal sinus, again in close association with lymphatic networks. Nasal lymphatics are connected to those of the ocular region via a lymphatic network along the nasolacrimal duct (NLD). By this means, lacrimal duct-associated lymphoid tissue (LDALT) has a dense supply with lymphatics.ConclusionsNALT and LDALT play a key role in the immune system of the mouse head, where they function as primary recognition sites for antigens. Using the dense lymphatic networks along the NLD described in this study, these antigens reach lymphatics near the palatine and are further drained to lymph nodes of the head and neck region.NALT and LDALT develop in immediate vicinity of lymphatic vessels. Therefore, we suggest a causative connection of lymphatic vessels and the development of lymphoid tissues.

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats.

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

MP72-20 CD31 ANTIBODY CONJUGATION IMPROVES RE-ENDOTHELIALIZATION OF ACELLULAR KIDNEY SCAFFOLDS FOR WHOLE KIDNEY ENGINEERING

You have accessJournal of UrologyTransplantation & Vascular Surgery II1 Apr 2014MP72-20 CD31 ANTIBODY CONJUGATION IMPROVES RE-ENDOTHELIALIZATION OF ACELLULAR KIDNEY SCAFFOLDS FOR WHOLE KIDNEY ENGINEERING In Kap Ko, Mehran Abolbashari, Jennifer Huling, Joao Zambon, Cheil Kim, John Jackson, Delrae Eckman, Anthony Atala, and James Yoo In Kap KoIn Kap Ko More articles by this author , Mehran AbolbashariMehran Abolbashari More articles by this author , Jennifer HulingJennifer Huling More articles by this author , Joao ZambonJoao Zambon More articles by this author , Cheil KimCheil Kim More articles by this author , John JacksonJohn Jackson More articles by this author , Delrae EckmanDelrae Eckman More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and James YooJames Yoo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2259AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The lack of transplantable allografts for renal failure has prompted the development of scaffold design and cell isolation techniques to re-create an engineered whole kidney. Decellularized solid organ xenografts such as the kidney hold great promise due to the preservation of structural integrity while removing all cellular components. However, acellular kidney vasculature needs an endothelial cell lining to avoid blood clotting and thrombus formation when transplanted into a living recipient. We developed a surface modification method to reinforce endothelial cell attachment onto renal vasculature via antibody conjugation, and evaluated the effectiveness of re-endothelialization in vivo through transplantation. METHODS Endothelial cell specific antibodies (CD31) were conjugated on the collagen pre-coated surfaces of microfluidic flow chamber to determine the levels of endothelial (MS1) cell attachment under various flow rates (20 to 100ml/hr). Decellularized porcine kidney scaffolds conjugated with CD31 antibodies were seeded with MS1 cells and preconditioned using a perfusion bioreactor. The re-endothelialized kidney scaffolds were transplanted in pigs. Angiographic and histological examinations were performed to determine the intactness of vascular trees, re-endothelialization of vasculatures, and platelet adhesion on vasculatures within the renal scaffold. RESULTS At all flow rates tested, there was no measurable detachment of MS1 cells in the antibody conjugated flow chambers. Angiography of the implanted kidneys showed improved vascular patency in all vessel sizes from the antibody treated scaffolds, as compared to the untreated scaffolds. This functional outcome was supported by the results of histological studies showing that CD31 antibody conjugation maintained the endothelialization of intermediate- and small- sized vasculatures, as well as preventing platelet adhesion. CONCLUSIONS Antibody conjugation-mediated MS1 cell attachment on the vasculature of implanted scaffolds improved vascular patency throughout the renal parenchyma. This functional outcome is supported by the maintenance of MS1 attachment and prevention of platelet adhesion on the endothelialized vasculatures during implantation. These results indicate that antibody conjugation improves re-endothelialization of vascular trees of bioengineered tissue, and this method could be used to engineer whole kidney. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e831 Advertisement Copyright & Permissions© 2014MetricsAuthor Information In Kap Ko More articles by this author Mehran Abolbashari More articles by this author Jennifer Huling More articles by this author Joao Zambon More articles by this author Cheil Kim More articles by this author John Jackson More articles by this author Delrae Eckman More articles by this author Anthony Atala More articles by this author James Yoo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

De novo development of circulating anti-endothelial cell antibodies rather than pre-existing antibodies is associated with post-transplant allograft rejection

Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.

Hyperacute Rejection of Living Related Kidney Grafts Caused by Endothelial Cell–Specific Antibodies: Case Reports

We describe two cases of hyperacute humoral rejection of living related kidney grafts despite negative pretransplantation T- and B-lymphocyte flow cytometric crossmatches and blood group identity. Retrospectively, antiendothelial IgG antibodies were detected on a panel of umbilical cord cells in the first case, and IgM antibodies against donor endothelial precursor cells were detected using a new endothelial cell crossmatch kit in the second case. Standard crossmatch methods using donor lymphocytes failed to detect these pathogenic antibodies and did not predict the danger of hyperacute rejection.

The endothelial cell-specific antibody PAL-E identifies a secreted form of vimentin in the blood vasculature.

During mammalian vascular development, endothelial cells form a complex array of vessels that differ markedly in structure and function, but the molecular basis for this vascular complexity is poorly understood. Recent insights into endothelial diversity have come from the identification of molecular markers expressed on distinct endothelial cell populations. One such marker, the PAL-E antibody, has been used for almost 20 years to distinguish blood and lymphatic vessels, but the identity of the protein recognized by PAL-E has been unknown. In the present study we have used protein purification and tandem mass spectrometry analysis of tryptic peptides to identify the PAL-E antigen as a secreted form of vimentin. Vimentin has been well characterized as an intracellular intermediate filament protein expressed broadly in mesenchymal cells. In contrast, PAL-E-reactive vimentin is secreted extracellularly, its synthesis is restricted to a distinct population of blood endothelial cells and activated macrophages, and PAL-E-reactive vimentin is found in circulating human blood. PAL-E-reactive vimentin does not arise from an endothelial cell-specific mRNA transcript but is the product of cell-specific posttranslational modification. The PAL-E antibody therefore defines secretion of vimentin as a molecular distinction among endothelial cells and exposes a novel, extracellular role for vimentin in the blood vasculature.

Microvascular structure after embolic focal cerebral ischemia in the rat

Objectives: We analyze morphological alterations of cerebral neovascularization after stroke using a new 3D imaging software program. Methods: Male Wistar rats underwent unilateral embolic middle cerebral artery occlusion (MCAo) by a single fibrin rich clot. Subjects were sacrificed from 1 to 28 days post infarct. Vessel perimeters were measured on coronal sections stained with endothelial cell-specific antibody to von Willebrand’s factor. Vessel segment lengths, diameters and number of vessels were analyzed on cerebral microvessels perfused with FITC-dextran 14 days after ischemia using LSCM and a 3-D vessel quantification program. Results: The mean number of microvessels with enlarged perimeters significantly increased in the ipsilateral cortex at day 7 when compared to the contralateral cortex (29.7±14.7 vs. 3.7±2.5, P<0.05). Subsequently, differences in the number of microvessels with enlarged perimeters decreased on days 14 and 28. Fourteen days post-MCA occlusion, microvessel segment length (15.0 vs. 26.0 μm, P<0.05) and diameter (3.14 vs. 3.75 μm, P<0.05) significantly decreased in the ipsilateral hemisphere when compared to the contralateral hemisphere, respectively. Furthermore, the mean total number of these smaller microvessels increased in the ipsilateral hemisphere (57.33±14.5 vs. 32.22±11.7, P<0.05). Conclusions: Focal cerebral ischemia induces morphological changes (early dilated microvessels followed by decreased microvessel segment length and diameter) that are consistent with newly generated microvessels.

Hypoxic preconditioning triggers myocardial angiogenesis: a novel approach to enhance contractile functional reserve in rat with myocardial infarction.

A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of "angiogens", mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore we sought to examine a novel method of stimulating myocardial angiogenesis through hypoxic preconditioning at both capillary (using anti-CD31) and arteriolar (using anti- alpha smooth muscle actin) levels and also investigate whether such treatments could preserve left ventricular contractile functional reserve and regional blood flow by increasing vascular endothelial growth factor (VEGF). Male Sprague-Dawley rats were randomly divided into four groups: normoxia+sham surgery (CS), normoxia+permanent left anterior descending coronary artery (LAD) occlusion (CMI), hypoxic preconditioning+sham surgery (HS) and hypoxic preconditioning+permanent LAD occlusion (HMI). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10+/-0.4% O(2)) for 4 h followed by a 24 h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia group were time matched with the preconditioned group and maintained under normoxic conditions for a 28 h period prior to LAD occlusion. Western blot analysis was performed to measure VEGF expression and TUNEL staining with endothelial cell-specific antibody, anti-VWF, was used to examine endothelial apoptosis. One, two and three weeks after the LAD occlusion, baseline left ventricular pressures were monitored and recorded. Pharmacological stress tests with dobutamine infusion in progressively increasing doses revealed significantly elevated contractile reserve at each dose point in the HMI group compared to the CMI group. The HMI group displayed statistically significant increases in capillary as well as arteriolar density after 1, 2 and 3 weeks post-operation. Blood flow was also significantly elevated in the HMI groups when compared to the CMI group. The extent of endothelial cell apoptosis was found to be inversely proportional to VEGF expression. It was concluded that hypoxic preconditioning stimulates myocardial angiogenesis to an extent sufficient to exert significant cardioprotection in a rat model of myocardial infarction progressing to heart failure as evidenced by increased capillary/arteriolar density and enhanced ventricular contractile functional reserve.

Effect of porosity on small-diameter vascular graft healing.

Experimental studies have reported that complete healing of small-diameter expanded polytetrafluoroethylene (ePTFE) grafts occurs only if the porosity of the graft is increased, thereby allowing ingrowth of perigraft capillaries yielding endothelial cells. This study investigates the effects of varied graft porosity on the healing characteristics of 2-mm internal diameter (ID) ePTFE grafts interposed in the rabbit common carotid artery. Four groups were evaluated: Group A (n = 8) standard (30-microm pores) ePTFE grafts; Group B (n = 8) increased porosity (60-microm pores) ePTFE grafts; Group C (n = 8) standard ePTFE; and Group D (n = 8) 60-microm ePTFE external graft surface was externally coated with an impermeable layer of polyurethane. Patency was 100% for all groups at 8 weeks. At explantation, the neointima was composed of primarily modified smooth muscle cells. Endothelial cells were only identified at the perianastomotic region using the endothelial cell-specific antibody CD31. The impermeable external polyurethane coating of ePTFE grafts had no effect on neointima formation, regardless of porosity.

Analysis of the sinusoidal endothelial cell organization during the developmental stage of the fetal rat liver using a sinusoidal endothelial cell specific antibody, SE-1.

The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membrane and was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid may start at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possibly regulated by the intimate association with hepatocytes.

Hyperacute rejections of two consecutive renal allografts and early loss of the third transplant caused by non-HLA antibodies specific for endothelial cells

The immunological rejection of HLA-identical kidney transplants indicates that non-HLA antigens may also be targets for transplant rejection. Interest in the possible role of endothelial specific antigens has grown steadily over the years. Most of the studies published, regarding the association of such antibodies with rejection, have demonstrated the reactivity of endothelial antibodies also with monocytes and keratinocytes, but not with lymphocytes. Such antibodies escape detection in conventional crossmatch tests. In this paper, we present a case report of a 10-year-old girl, whose two consecutive kidney allografts, (one living and one cadaveric donor) were hyperacutely rejected in spite of the fact that she had neither been alloimmunized, nor had any HLA-specific antibodies. Endothelial cell specific antibodies were detected in vivo and in vitro after transplantation only 11 days apart, which were considered to be responsible for rejection. The third cadaveric kidney was lost within 1 week post-transplant. Immunopathological investigation of the three rejected grafts revealed deposition of IgM in the endothelium of arteries and in some glomeruli. No deposition of IgG antibodies was found. Antibodies from this patient did not react with lymphocytes, monocytes or keratinocytes. Patient serum had IgM antibodies that were specifically reactive with cultured endothelial cells, demonstrated by binding in vitro and by complement-dependent cytotoxicity of IL-β stimulated endothelial cells. No HLA antibodies were found following the first two transplantations, but were demonstrated 1 week after the third transplantation, at the time of an acute irreversible rejection. Western blots of proteins solubilized from endothelial cell membranes, indicated that the antibodies reacted with a 97–110 kD protein. Endothelial cell antigen preparations were made from several different umbilical cord veins. Some primary cell cultures, but not all, reacted with the patient's serum. Therefore, we suggest that the target determinant might be polymorphic. These findings imply that the non-HLA endothelial cell specific molecules may function as target(s) for hyperacute antibody-mediated destruction of kidney allografts.

Restoring and immunohistological examination of Feulgen stained avian embryonic tissues using iron hematoxylin and endothelial cell specific antibody.

The immunohistological method described here permits re-examination of previously Feulgen stained quail-chick chimera tissues for vascular development using the monoclonal antibody QH1 which specifically recognizes quail hemangioblastic cells. Weigert's iron hematoxylin has been used to restore faded or bleached Feulgen stained chimeric avian tissues. Species-specific differences in nuclear morphology are as evident with iron hematoxylin staining as they are with Feulgen staining.

Immunocytochemistry of Angiosarcomas: A Study of 19 Cases with Special Emphasis on the Applicability of Endothelial Cell Specific Markers to Routinely Prepared Tissues

Since the advent of endothelial cell specific antibodies, immunocytochemistry has become an increasingly used tool in the diagnosis of malignant endothelial tumors or tumors of an alleged vascular nature. In surgical pathology the most frequently applied markers are anti-Factor VIII-related antigen (anti-FVIII-R:AG) and Ulex europeus I agglutinin (UEA I) because of their ability to work on routinely formalin-fixed paraffin-embedded tissue. However, these markers label angiosarcomas inconstantly, which has prompted the search for more reliable markers, especially monoclonal antibodies specific for endothelial cell specific antigens. BMA 120 (formerly designated BW 200) is an endothelial cell specific monoclonal antibody detecting an epitope of a formalin resistant antigen. This marker labels a considerable number of angiosarcoma cells and it was compared with the staining patterns of anti-FVIIIR:Ag and UEA I in 19 angiosarcomas. Special regard was given to the distribution of antibodies in different parts of angiosarcomas.