IgA Anti-ß2-Glycoprotein I Antibodies as Markers of Thrombosis and Severity in COVID-19 Patients.

Patients with COVID-19 may develop a hypercoagulable state due to tissue and endothelial injury, produced by an unbalanced immune response. Therefore, an increased number of thromboembolic events has been reported in these patients. The aim of this study is to investigate the presence of antiphospholipid antibodies (aPL) in COVID-19 patients, their role in the development of thrombosis and their relationship with the severity of the disease. In this retrospective study, serum samples from 159 COVID-19 patients and 80 healthy donors were analysed for the presence of aPL. A total of 29 patients (18.2%) and 14 healthy donors (17.5%) were positive for aPL. Nineteen COVID-19 patients (12%) but no healthy donor presented a positive percentage of the IgA isotype aPL. IgA anti-ß2-glycoprotein I antibodies (anti-ß2GPI) were the most frequent type (6.3%) in patients but was not detected in any healthy donor. The positivity of this antibody was found to be significantly elevated in patients with thromboembolic events (25% vs. 5%, p = 0.029); in fact, patients with positive IgA anti-ß2GPI had an incidence of thrombosis over six times higher than those who had normal antibody concentrations [OR (CI 95%) of 6.67 (1.5-30.2), p = 0.014]. Additionally, patients with moderate-severe disease presented a higher aPL positivity than patients with mild disease according to the Brescia (p = 0.029) and CURB-65 (p = 0.011) severity scales. A multivariate analysis showed that positivity for IgA anti-ß2GPI is significantly associated with disease severity measured by CURB-65 [OR (CI 95%) 17.8 (1.7-187), p = 0.0016]. In conclusion, COVID-19 patients have a significantly higher positive percentage of the IgA isotype aPL than healthy donors. IgA anti-ß2GPI antibodies were the most frequently detected aPL in COVID-19 patients and were associated with thrombosis and severe COVID-19 and are thus proposed as a possible marker to identify high-risk patients.

Kinking graft-an exceptional late complication of axillofemoral bypass grafting.

The axillary-femoral bypass is an extra-anatomical arterial reconstruction technique whose indications and complications have been thoroughly discussed in the literature. Shortening or lengthening of the prosthesis (by axillary artery traction or graft angulation, respectively) as a late postoperative complication of the procedure has been described only exceptionally. Here we report a kinking of the prosthesis with a very illustrative figure.

VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.

The angiogenesis inhibitor thrombospondin-1 inhibits acute cutaneous hypersensitivity reactions.

There is increasing evidence that vascular remodeling and endothelial cell activation promote acute and chronic inflammation. Thrombospondin 1 (TSP-1) is a potent endogenous angiogenesis inhibitor thought to play an important role in maintaining cutaneous vascular quiescence. We first investigated TSP-1 expression in human and contact hypersensitivity (CHS) reactions and found that TSP-1 was upregulated in the inflamed skin of patients and in mice. To elucidate the function of TSP-1 in cutaneous inflammation, we induced CHS reactions in the skin of mice with targeted epidermal TSP-1 overexpression in TSP-1-deficient mice and in wild-type mice. We found decreased edema formation, angiogenesis, and inflammatory infiltrate in the inflamed skin of TSP-1 transgenic mice. Conversely, TSP-1-deficient mice exhibited an enhanced and prolonged inflammation, characterized by increased edema formation, enhanced vascular remodeling, and increased neutrophilic infiltrate, when compared with wild-type mice. Moreover, we found strong upregulation of the proinflammatory cytokines IL-1beta, macrophage inflammatory protein 2, and tumor necrosis factor-alpha in the inflamed skin of TSP-1-deficient mice. Our results indicate that TSP-1 downregulates cutaneous delayed-type hypersensitivity reactions by acting on several distinct pathways mediating skin inflammation.

Novel anti-inflammatory properties of the angiogenesis inhibitor vasostatin.

Endothelial cells are critically involved in the pathogenesis of inflammation, which is characterized by vasopermeability, plasma leakage, leukocyte recruitment, and neovascularization. Therefore, inhibitors of endothelial cell function could reduce inflammation. In this study, we evaluated the effects of the angiogenesis inhibitor vasostatin on inflammations induced by contact hypersensitivity reactions in mouse ears. Vasostatin-treated mice revealed significantly reduced edema formation, resulting from lower plasma leakage and inhibition of inflammation-associated vascular remodeling. Intravital microscopy studies of inflamed ears showed a decrease in the fraction of rolling leukocytes in vasostatin-treated mice, and Lycopersicon esculentum lectin-perfused ears revealed fewer leukocytes adherent to the vessel wall. The inflammatory infiltrate from vasostatin-treated mice was characterized by fewer CD8+ T cells, neutrophils, and macrophages compared to the saline-treated animals. In a modified Miles assay, vasostatin inhibited vascular endothelial growth factor-A-induced permeability, and inflamed ear tissues from vasostatin-treated mice expressed significantly reduced levels of the vascular destabilizer angiopoietin-2. These results reveal a previously unrecognized anti-inflammatory property of the angiogenesis inhibitor vasostatin, and suggest that vasostatin is a potential candidate drug for the treatment of inflammation.

Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes.

Cutaneous melanoma is a common melanocytic neoplasm that can quickly metastasize to regional lymph nodes. Currently, prognosis is determined by measuring tumor thickness but more reliable markers for metastatic spread are urgently needed. We investigated whether the extent of tumor lymphangiogenesis can predict melanoma metastasis to sentinel lymph nodes. We quantified the extent of tumor lymphangiogenesis, as well as other factors, in excised primary tumors and in sentinel lymph node biopsy samples from 45 patients with primary cutaneous melanoma. The results were correlated with histological and clinical outcome. Primary melanomas from patients whose tumors had metastasized to the sentinel lymph nodes contained prominent 'hot spots' of increased lymphatic vessel density, compared to nonmetastatic tumors. Multivariate risk analysis revealed that the lymphatic vascular area of primary melanomas, an index of tumor lymphangiogenesis, was the most sensitive prognostic marker for sentinel lymph node metastasis, and was even able to more accurately predict which tumors were metastatic to sentinel lymph nodes than the currently used method of measuring tumor thickness. Highly lymphangiogenic melanomas maintained their lymphangiogenic activity after metastasis to the sentinel lymph node. The extent of tumor lymphangiogenesis is a highly sensitive (83%) and specific (89%) prognostic marker of lymph node metastasis. Assessment of lymphangiogenesis in primary melanomas may be a more effective approach than the currently used technique of measuring tumor thickness in selecting patients with early metastatic disease for aggressive therapy.

Molecular characterization of lymphatic endothelial cells.

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.

A critical role of placental growth factor in the induction of inflammation and edema formation.

Angiogenesis is a prominent feature of a number of inflammatory human diseases, including rheumatoid arthritis, psoriasis, and cutaneous delayed-type hypersensitivity (DTH) reactions. Up-regulation of placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has been found in several conditions associated with pathologic angiogenesis; however, its distinct role in the control of angiogenesis has remained unclear. To directly investigate the biologic function of PlGF in cutaneous inflammation and angiogenesis, DTH reactions were investigated in the ear skin of wild-type mice, of PlGF-deficient mice, and of transgenic mice with targeted overexpression of human PlGF-2 in epidermal keratinocytes, driven by a keratin 14 promoter expression construct. Chronic transgenic delivery of PlGF-2 to murine epidermis resulted in a significantly increased inflammatory response, associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration than seen in wild-type mice. Conversely, PlGF deficiency resulted in a diminished and abbreviated inflammatory response, together with a reduction of inflammatory angiogenesis and edema formation. VEGF expression was up-regulated at a comparable level in the inflamed skin of all genotypes. These findings reveal that placental growth factor plays a critical role in the control of cutaneous inflammation, and they suggest inhibition of PlGF bioactivity as a potential new approach for anti-inflammatory therapy.

Increased and prolonged inflammation and angiogenesis in delayed-type hypersensitivity reactions elicited in the skin of thrombospondin-2--deficient mice.

Angiogenesis and enhanced microvascular permeability are hallmarks of a large number of inflammatory diseases. Although up-regulation of proangiogenic factors such as vascular endothelial growth factor and interleukin-8 have been previously reported in inflamed tissue, the biologic role of endogenous inhibitors of angiogenesis in inflammation has remained unclear. To investigate the biologic role of the potent angiogenesis inhibitor thrombospondin-2 (TSP-2) in the control of cutaneous inflammation, delayed-type hypersensitivity reactions were elicited in the ear skin of wild-type and TSP-2-deficient mice by topical sensitization and challenge with oxazolone. Cutaneous TSP-2 expression was up-regulated in the inflamed skin of wild-type mice, predominantly in dermal fibroblasts and microvessels. Lack of TSP-2 resulted in a significantly enhanced inflammatory response with increased angiogenesis, edema formation, and inflammatory infiltration. Ear swelling and inflammation persisted for more than 2 weeks in TSP-2-deficient mice, as compared with 1 week in wild-type mice. Although baseline vascular permeability was unchanged, significantly enhanced microvascular leakage was found in the inflamed skin of TSP-2-deficient mice. Moreover, the fraction of rolling leukocytes was significantly increased in the untreated skin of TSP-2-deficient mice. These results reveal an important role of TSP-2 in limiting the extent and the duration of edema formation, angiogenesis, and inflammatory cell infiltration during acute and chronic inflammation.

Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth.

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by alpha-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.

Vascular endothelial growth factor-A (VEGF-A) expression is up-regulated in several inflammatory diseases including psoriasis, delayed-type hypersensitivity (DTH) reactions, and rheumatoid arthritis. To directly characterize the biologic function of VEGF-A in inflammation, we evaluated experimental DTH reactions induced in the ear skin of transgenic mice that overexpress VEGF-A specifically in the epidermis. VEGF-A transgenic mice underwent a significantly increased inflammatory response that persisted for more than 1 month, whereas inflammation returned to baseline levels within 7 days in wild-type mice. Inflammatory lesions in VEGF-A transgenic mice closely resembled human psoriasis and were characterized by epidermal hyperplasia, impaired epidermal differentiation, and accumulation of dermal CD4+ T-lymphocytes and epidermal CD8+ lymphocytes. Surprisingly, VEGF-A also promoted lymphatic vessel proliferation and enlargement, which might contribute to the increased inflammatory response, as lymphatic vessel enlargement was also detected in human psoriatic skin lesions. Combined systemic treatment with blocking antibodies against VEGF receptor-1 (VEGFR-1) and VEGFR-2 potently inhibited inflammation and also decreased lymphatic vessel size. Together, these findings reveal a central role of VEGF-A in promoting lymphatic enlargement, vascular hyperpermeability, and leukocyte recruitment, thereby leading to persistent chronic inflammation. They also indicate that inhibition of VEGF-A bioactivity might be a new approach to anti-inflammatory therapy.