A clinicopathological description of COVID-19-induced chilblains (COVID-toes) correlated with a published literature review.

BACKGROUND: The abundance of publications of COVID-19-induced chilblains has resulted in a confusing situation. METHODS: This is a prospective single-institution study from 15 March to 13 May 2020. Thirty-two patients received PCR nasopharyngeal swabs. Of these, 28 patients had a thoracic CT-scan, 31 patients had blood and urine examinations, 24 patients had skin biopsies including immunohistochemical and direct immunofluorescence studies, and four patients had electron microscopy. RESULTS: COVID-19-induced chilblains are clinically and histopathologically identical to chilblains from other causes. Although intravascular thrombi are sometimes observed, no patient had a systemic coagulopathy or severe clinical course. The exhaustive clinical, radiological, and laboratory work-up in this study ruled-out other primary and secondary causes. Electron microscopy revealed rare, probable viral particles whose core and spikes measured from 120 to 133 nm within endothelium and eccrine glands in two cases. CONCLUSION: This study provides further clinicopathologic evidence of COVID-19-related chilblains. Negative PCR and antibody tests do not rule-out infection. Chilblains represent a good prognosis, occurring later in the disease course. No systemic coagulopathy was identified in any patient. Patients presenting with acral lesions should be isolated, and chilblains should be distinguished from thrombotic lesions (livedo racemosa, retiform purpura, or ischemic acral necrosis).

Prolonged Cardiopulmonary Bypass is Associated With Endothelial Glycocalyx Degradation.

BACKGROUND: The endothelial glycocalyx (EG) is involved in critical regulatory mechanisms that maintain endothelial vascular integrity. We hypothesized that prolonged cardiopulmonary bypass (CPB) may be associated with EG degradation. We performed an analysis of soluble syndecan-1 levels in relation to duration of CPB, as well as factors associated with cell stress and damage, such as mitochondrial DNA (mtDNA) and inflammation. METHODS: Blood samples from subjects undergoing cardiac surgery with CPB (n = 54) were obtained before and during surgery, 4-8 h and 24 h after completion of CPB, and on postoperative day 4. Flow cytometry was used to determine subpopulations of white blood cells. Plasma levels of mtDNA were determined using quantitative polymerase chain reaction and plasma content of shed syndecan-1 was measured. To determine whether syndecan-1 was signaling white blood cells, the effect of recombinant syndecan-1 on mobilization of neutrophils from bone marrow was tested in mice. RESULTS: CPB is associated with increased mtDNA during surgery, increased syndecan-1 blood levels at 4-8 h, and increased white blood cell count at 4-8 h and 24 h. Correlation analysis revealed significant positive associations between time on CPB and syndecan-1 (rs = 0.488, P < 0.001) and level of syndecan-1 and neutrophil count (rs = 0.351, P = 0.038) at 4-8 h. Intravenous administration of recombinant syndecan-1 in mice resulted in a 2.5-fold increase in the number of circulating neutrophils, concurrent with decreased bone marrow neutrophil number. CONCLUSIONS: Longer duration of CPB is associated with increased plasma levels of soluble syndecan-1, a signal for EG degradation, which can induce neutrophil egress from the bone marrow. Development of therapy targeting EG shedding may be beneficial in patients with prolonged CPB.

Endothelial alterations in a canine model of immune thrombocytopenia.

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.

Sinusoidal obstruction syndrome (SOS): A light and electron microscopy study in human liver.

AIMS: Oxaliplatin is an important chemotherapeutic agent, used in the treatment of hepatic colorectal metastases, and known to induce the sinusoidal obstruction syndrome (SOS). Pathophysiological knowledge concerning SOS is based on a rat model. Therefore, the aim was to perform a comprehensive study of the features of human SOS, using both light microscopy (LM) and electron microscopy (EM). METHODS AND RESULTS: Included were all patients of whom wedge liver biopsies were collected during a partial hepatectomy for colorectal liver metastases, in a 4-year period. The wedge biopsy were perfusion fixated and processed for LM and EM. The SOS lesions were selected by LM and details were studied using EM. Material was available of 30 patients, of whom 28 patients received neo-adjuvant oxaliplatin. Eighteen (64%) of the 28 patients showed SOS lesions, based on microscopy. The lesions consisted of sinusoidal endothelial cell detachment from the space of Disse on EM. In the enlarged space of Disse a variable amount of erythrocytes were located. CONCLUSION: Sinusoidal endothelial cell detachment was present in human SOS, accompanied by enlargement of the space of Disse and erythrocytes in this area. These findings, originally described in a rat model, were now for the first time confirmed in human livers under clinically relevant settings.

Positive C4d staining of the portal vein endothelium in the liver of patients with biliary atresia: a role of humoral immunity in ongoing liver fibrosis.

PURPOSE: This study aimed to clarify the role of complement activation in fibrogenesis in BA. METHODS: In total, 27 paraffin-embedded liver biopsy samples were immunohistochemically analyzed using C4d polyclonal antibody, vascular cell adhesion molecule-1 (VCAM-1), and CD45. The liver samples were obtained from 25 patients during Kasai operation, and two additional specimens were obtained from 2 patients by needle biopsy later at the time of liver function deterioration. The degree of liver fibrosis was histologically graded 1-3. RESULTS: Among the 25 samples, 9 showed C4d-positive immunostaining localized on the endothelia of a few portal veins in the portal tract. The degree of fibrosis was correlated with C4d staining (p = 0.025). The age at Kasai operation correlated with the degree of fibrosis and the C4d positivity. Two needle biopsy samples were positive for C4d. Among 13 samples submitted for VCAM-1 staining, 2 negative samples were C4d negative and all positive C4d samples were VCAM-1 positive with CD45 mononuclear cell infiltration. CONCLUSION: These findings suggest that ongoing cirrhosis could be a result of progressive "vasculopathy" of the portal vein caused by humoral and cell-mediated immune interaction.

Nanomechanics of the endothelial glycocalyx in experimental sepsis.

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.

Does carbon dioxide pneumoperitoneum altering pressure levels lead to ultrastructural damage of fallopian tube and ovary?

AIM: To assess carbon dioxide pneumoperitoneum and its different pressure levels related to cellular injury on ovarian surface epithelium, endothelium, and fallopian tube ciliated epithelium in laparoscopic rat model. MATERIALS AND METHODS: Twenty-four Wistar-Albino female rats were randomized into three groups. Laparotomy was applied for Group 1 (control). Groups 2 and 3 had laparoscopy with pneumoperitoneum pressures at 10 mmHg and 15 mmHg, respectively. After 150 minutes (last 30 minutes was after desufflation for Group 2 and 3) in all groups, bilateral ovariectomy and salpingectomy were performed. The ultrastructures of ovarian surface epithelium, ovarian endothelium, and fallopian tube ciliated epithelium were evaluated by transmission electron microscope. Ovarian surface epithelium changes were divided into three groups, apical surface changes, lateral surface chances, and organelle modification/damage. RESULTS: No apical or lateral surface changes or organelle modifications in ovarian surface epithelium were observed in the control group. Apical ovarian surface epithelium changes were statistically significant in Groups 2 and 3 in comparison to the control group. No significant differences were observed with regards to lateral surface changes in all groups. The organelle modification was only significant in Group 3 compared to the control group. The authors revealed that the ultrastructures of the ovarian endothelium and fallopian tube epithelium were not affected by pneumoperitoneum. CONCLUSIONS: Pneumoperitoneum may cause ischemia-reperfusion damage in ovarian cortex correlated with the amount of pressure.

Adipose-derived stem cells (ASCs) as a source of endothelial cells in the reconstruction of endothelialized skin equivalents.

Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models.

Histopathology of the trabecular meshwork and Schlemm's canal in primary angle-closure glaucoma.

PURPOSE: To investigate the detailed histopathology of trabecular meshwork changes associated with primary angle closure glaucoma (PACG). METHODS: Thirty trabecular blocks obtained from trabeculectomy (TLE) of 25 PACG patients were embedded in paraffin for immunohistochemical staining of thrombomodulin, CD68, D2-40, and epon for transmission electron microscopy. Eleven TLE blocks obtained from normal-tension glaucoma patients were used as a control. Histologic changes of outflow routes were analyzed by comparing the existence of iridotomy, gonioscopy-evaluated angle closure, intraocular pressure (IOP), episodes of acute attack, visual field defect classified by Aulhorn-Greve, anterior chamber depth, lens thickness, and axial length. RESULTS: Occlusion of the Schlemm's canal (SC) of <150 µm was observed in 11 eyes, which significantly correlated with gonioscopy-evaluated angle closure (T = 19.33 > χ² (f,α) = 9.488). Moderate correlation between SC occlusion and IOP before TLE was also observed (correlation coefficient: -0.540). Slightly negative or no correlations were found between SC occlusion and the other parameters. Thinned SC endothelium at the junction or degenerated SC endothelium and various degrees of SC occlusion and fusion of the trabecular beams where trabecular cells degenerated with damaged mitochondria were the general findings in the PACG eyes involved in this study. CONCLUSIONS: Persistent trabecular-iris contact or peripheral anterior synechia may block aqueous outflow resulting in a progressive process of SC endothelial damage and subsequent SC occlusion, as well as trabecular cell damage possibly due to impairment of mitochondrial function and subsequent fusion of the trabecular beams. These changes may be the reason for residual glaucoma after laser iridotomy or cataract surgery.

Effect of preischemic treatment with fenofibrate, a peroxisome proliferator-activated receptor-α ligand, on hepatic ischemia-reperfusion injury in rats.

Liver ischemia/reperfusion (I/R) injury is a serious clinical problem. The reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) are important mediators in liver I/R injury. This study was designed to investigate the effect of preischemic treatment with fenofibrate (Peroxisome proliferator-activated receptor- α agonist) on the oxidative stress and inflammatory response to hepatic I/R injury in rats. Hepatic I/R was induced by clamping the blood supply of the left lateral and median lobes of the liver for 60 min, followed by reperfusion for 4 h. Each animal group was pretreated with a single dose of fenofibrate (50 mg/kg body weight) intraperitoneally 1 h before ischemia. At the end of reperfusion, blood samples and liver tissues were obtained to assess serum alanine aminotransferase (ALT), TNF-α, hepatic malondialdehyde (MDA) and superoxide dismutase activity (SOD). Liver specimens were obtained and processed for light and electron microscopic study. Hepatic I/R induced a significant elevation of serum ALT and TNF-α with significant elevation of hepatic MDA and reduction of SOD activity. Histopathological examination revealed hepatic inflammation, necrosis and apoptosis. Preischemic treatment with fenofibrate at a dose of 50 mg/kg significantly attenuated the biochemical and structural alterations of I/R-induced liver injury.

Review: molecular pathogenesis of blood-brain barrier breakdown in acute brain injury.

Historically, the blood-brain barrier (BBB) was considered to be at the level of cerebral endothelium. Currently, the interaction of endothelium with other components of the vessel wall and with neurones and glial cells is considered to constitute a functional unit, termed the neurovascular unit that maintains cerebral homeostasis in steady states and brain injury. The emphasis of this review is on cerebral endothelium, the best-studied component of the neurovascular unit, and its permeability mechanisms in health and acute brain injury. Major advances have been made in unravelling the molecular structure of caveolae and tight junctions, both of which are components of the structural barrier to the entry of plasma proteins into brain. Time course studies suggest that caveolar changes precede junctional changes in acute brain injury. Additional factors modulating BBB permeability in acute brain injury are matrix metalloproteinases-2 and 9 and angiogenic factors, the most notable being vascular endothelial growth factor-A and angiopoietins (Ang) 1 and 2. Vascular endothelial growth factor-A and Ang2 have emerged as potent inducers of BBB breakdown while Ang1 is a potent anti-leakage factor. These factors have the potential to modulate permeability in acute brain injury and this is an area of ongoing research. Overall, a combination of haemodynamic, structural and molecular alterations affecting brain endothelium results in BBB breakdown in acute brain injury.

Absence of the adenosine A2A receptor confers pulmonary arterial hypertension and increased pulmonary vascular remodeling in mice.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by sustained elevation of pulmonary vascular resistance resulting from endothelial and smooth muscle cell dysfunction and collagen deposition in pulmonary vascular walls. In this study, we investigated the role of the adenosine A(2A) receptor (A(2A)R) in the development of PAH by determining the effect of genetic inactivation of A(2A)Rs on pulmonary vascular remodeling in mice. METHODS AND RESULTS: We characterized hemodynamic, histological and ultrastructural changes in pulmonary vascular remodeling in A(2A)R knockout (KO) mice compared with their wild-type (WT) littermates after exposure to normoxia and hypoxic conditions. After exposure to normoxia, compared to WT mice, A(2A)R KO mice displayed: (1) increased right ventricular systolic pressures and an elevated ratio of the right ventricle over left ventricle plus septum (Fulton index), (2) increased wall area and thickness as well as enhanced smooth muscle actin immunoreactivity in pulmonary resistance vessels, (3) increased proliferating cell nuclear antigen-positive cells in pulmonary resistance vessels and (4) increased smooth muscle cells hypertrophy and collagen deposition in the adventitia of pulmonary arteriole walls as revealed by electron microscope. By contrast, histological analysis revealed no features of hypertensive nephropathy in A(2A)R KO mice and there was no significant difference in systemic blood pressure, and left ventricular masses among the 3 genotypes. Furthermore, following chronic exposure to hypoxia, A(2A)R KO mice exhibited exacerbated elevation in right ventricular systolic pressure, hypertrophy of pulmonary resistance vessels and increased cell proliferation in pulmonary resistance vessels, compared to WT littermates. Thus, genetic inactivation of A(2A)Rs selectively produced PAH and associated increased smooth muscle proliferation and collagen deposition. CONCLUSIONS: Extracellular adenosine acting at A(2A)Rs represents an important regulatory mechanism to control the development of PAH and pulmonary vascular remodeling.

p130Cas, Crk-associated substrate plays essential roles in liver development by regulating sinusoidal endothelial cell fenestration.

UNLABELLED: p130Cas, Crk-associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously showed that mice in which Cas was deleted (Cas(-/-)) died in utero because of early cardiovascular maldevelopment. To further investigate the in vivo roles of Cas, we generated mice with a hypomorphic Cas allele lacking the exon 2-derived region (Cas(Deltaex2/Deltaex2)), which encodes Src homology domain 3 (SH3) of Cas. Cas(Deltaex2/Deltaex2) mice again died as embryos, but they particularly showed progressive liver degeneration with hepatocyte apoptosis. Because Cas expression in the liver is preferentially detected in sinusoidal endothelial cells (SECs), the observed hepatocyte apoptosis was most likely ascribable to impaired function of SECs. To address this possibility, we stably introduced a Cas mutant lacking the SH3 domain (Cas DeltaSH3) into an SEC line (NP31). Intriguingly, the introduction of Cas DeltaSH3 induced a loss of fenestrae, the characteristic cell-penetrating pores in SECs that serve as a critical route for supplying oxygen and nutrients to hepatocytes. The disappearance of fenestrae in Cas DeltaSH3-expressing cells was associated with an attenuation of actin stress fiber formation, a marked reduction in tyrosine phosphorylation of Cas, and defective binding of Cas to CrkII. CONCLUSION: Cas plays pivotal roles in liver development through the reorganization of the actin cytoskeleton and formation of fenestrae in SECs.

Ultrastructural modification of the endothelium in placental insufficiency and microangiopathies.

Structural reorganization of endotheliocytes was studied on models of various pathological processes: placental dysfunction, glomerular pathology, vibration syndrome, antiphospholipid syndrome, and diffuse angiokeratoma, all of these characterized by endothelial insufficiency. Universal modification of endothelial associations was revealed. It included a chain of stereotypical reactions: from degeneration alternating with compensatory hypertrophy to subsequent atrophy and death of endotheliocytes. The time course of the process was confirmed by the results of light microscopy in combination with ultrastructural examination and by evaluation of the biosynthetic reactions by in vitro radioautography.

Extracellular histones are major mediators of death in sepsis.

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture.

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (-8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as -5 to -3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased over time. QRT-PCR analysis of the anti-CEACAM1 treated ES cells revealed a significant decrease in the expression of Ceacam1, Pecam1, Tie-1, and Flk-1, while VE-Cad and Tie-2 expression were unaffected. These results suggest that the expression and signaling of CEACAM1 may affect the expression of other factors known to play critical roles in vasculogenesis. Furthermore this 3D model of vasculogenesis in an environment of extracellular matrix may be a useful model for comparison to existing models of angiogenesis.

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Nano-surgery at the leukocyte-endothelial docking site.

The endothelium has an important role in controlling the extravasation of leukocytes from blood to tissues. Endothelial permeability for leukocytes is influenced by transmembrane proteins that control inter-endothelial adhesion, as well as steps of the leukocyte transmigration process. In a cascade consisting of leukocyte rolling, adhesion, firm adhesion, and diapedesis, a new step was recently introduced, the formation of a docking structure or "transmigratory cup." Both terms describe a structure formed by endothelial pseudopods embracing the leukocyte. It has been found associated with both para- and transcellular diapedesis. The aim of this study was to characterize the leukocyte-endothelial contact area in terms of morphology and cell mechanics to investigate how the endothelial cytoskeleton reorganizes to engulf the leukocyte. We used atomic force microscopy (AFM) to selectively remove the leukocyte and then analyze the underlying cell at this specific spot. Firmly attached leukocytes could be removed by AFM nanomanipulation. In few cases, this exposed 8-12 microm wide and 1 microm deep footprints, representing the cup-like docking structure. Some of them were located near endothelial cell junctions. The interaction area did not exhibit significant alterations neither morphologically nor mechanically as compared to the surrounding cell surface. In conclusion, the endothelial invagination is formed without a net depolymerization of f-actin, as endothelial softening at the site of adhesion does not seem to be involved. Moreover, there were no cases of phagocytotic engulfment, but instead the formation of a transmigratory channel could be observed.

Expression of dicarbonyl/L-xylulose reductase (DCXR) in human skin and melanocytic lesions: morphological studies supporting cell adhesion function of DCXR.

BACKGROUND: It has been proposed that dicarbonyl/L-xylulose reductase (DCXR) is increased in prostate cancer. Also, when compared with normal skin, a virtual northern blot shows increased expression of DCXR in melanomas. METHODS: We investigated DCXR expression in a tissue microarray, with 20 benign and 33 malignant melanocytic lesions and possible colocalization of DCXR with cell adhesion molecules using double immunofluorescence/confocal microscopy in normal human skin. RESULTS: Most nevi expressed DCXR in the cytoplasmic membrane, but some melanomas (20-30%) showed loss of membranous expression with inappropriate cytoplasmic or nuclear expression. Perinuclear Golgi expression was found in primary (14%) and metastatic (32%) melanomas showing dishesive growth pattern. Overall, the intensity of expression was stronger in nevi compared with melanomas (p < 0.005). In normal skin, DCXR was colocalized with E-cadherin and beta-catenin at the intercellular membranes of keratinocytes and with CD31 at the intercellular junctions of endothelial cells. DCXR was localized in the cytoplasmic membrane of normal melanocytes. CONCLUSIONS: These findings indicate that decreased membranous expression of DCXR with altered subcellular localization appears to be associated with malignant progression of melanocytic lesions. We show for the first time the expression of DCXR in normal keratinocytes, melanocytes and endothelial cells.