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Background: SARS-CoV-2 infection may lead to myocardial and endothelial damage. The present study sought to characterize the cardiovascular sequel in a large group of consecutive patients admitted for out-patient cardiovascular follow-up after a symptomatic COVID-19 infection., Methods: The aims of this study were as follows: to evaluate the presence of post-covid cardiovascular symptoms in an unselected population of outpatients referred to a post-COVID outpatient cardiology clinic and to characterize the long-term abnormalities associated with a more severe COVID-19 infection clinical course. A total of 914 patients were included in this single-center, observational, cross-sectional study, of which 163 were hospitalized and 149 required mechanical ventilation for COVID-19 pneumonia. Patients were analyzed at follow-up according to the care setting during the initial presentation., Results: The median time to follow-up was 126 days. At that time, only 3.5% of patients reported no persistent dyspnea, chest pain, or fatigue on exertion. In a follow-up echocardiographic assessment, patients who required hospitalization showed slight alterations in the pulmonary acceleration time and the tricuspid regurgitation pressure gradient, as well as reduced exercise tolerance during treadmill exercise testing when compared to patients with a benign clinical course. 24-hour Holter EKG monitoring or 24-hour blood pressure monitoring did not identify significant differences between the analyzed subgroups., Conclusions: The current study reports on an association between COVID-19 severity and the presence of cardiovascular alterations at follow-up. A simple diagnostic protocol, comprising an exercise treadmill test and transthoracic echocardiography is useful in identifying patients who may benefit from regular, structured cardiovascular medical care.

Background: Okra pods contain heat-sensitive substances, such as phenolic compounds and other phytochemicals that can be degraded when okra pods are subjected to heat treatment. The understanding of the impact of high humidity hot air impingement blanching (HHAIB) on the changes in physicochemical properties of polysaccharides and phytochemicals of okra pods is of great importance because over-blanching may result in cell membrane disruption and changes in biologically active compounds under prolonged exposure to the thermal treatment. Therefore, the present study aimed to investigate the effect of HHAIB on the changes in physicochemical properties of pectins and phytochemicals extracted from okra pods., Results: Both the HHAIB time and method of extraction influenced their physicochemical characteristics and biological activity. Pectin fractions subjected to HHAIB were composed of polygalacturonic acid, rhamnogalacturonan, glucomannan, galactan, mannose, arabinose, rhamnose, calcium pectate and arabinogalactan. The contents of total phenolics, total flavonoids and antioxidant activity of extracts mostly increased during HHAIB (i.e. up to 19.0%, 13.2% and 35.3%, respectively). However, HHAIB reduced the chlorophyll-a (up to 55.7%) and lycopene (up to 52.6%) contents of okra pods., Conclusion: The acquired knowledge may be useful for better understanding and optimization of technologies based on HHAIB treatment. The HHAIB treated okra can be a promising natural alternative in different applications, including its use as a replacement of some ingredients in food or non-food systems as a result of richness in polysaccharides and polyphenols, as well as high antioxidant properties. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

In the present study we described the role of alpha9beta1 integrin in glioblastoma progression following its interaction with nerve growth factor (NGF). The level of expression of alpha9beta1 on astrocytomas is correlated with increased grade of this brain tumor and is highest on glioblastoma, whereas normal astrocytes do not express this integrin. Two glioblastoma cell lines, LN229 and LN18, that are alpha9beta1 integrin positive and negative, respectively, were used for alpha9beta1 integrin-dependent NGF-induced tumor progression. NGF was a significant promoter of promigratory and pro-proliferative activities of glioblastoma cells through direct interaction with alpha9beta1 integrin and activation of MAPK Erk1/2 pathway. The level of NGF increases approximately threefold in the most malignant glioma tissue when compared with normal brain. This increase is related to secretion of NGF by tumor cells. Specific inhibitors of alpha9beta1 integrin or gene silencing inhibited NGF-induced proliferation of LN229 cell line to the level shown by LN18 cells. VLO5 promoted alpha9beta1-dependent programmed cell death by induction of intrinsic apoptosis pathway in cancer cells. LN229 cells were rescued from proapoptotic effect of VLO5 by the presence of NGF. This disintegrin significantly inhibited tumor growth induced by implantation of LN229 cells to the chorioallantoic membrane (CAM) of quail embryonic model, and this inhibitory effect was significantly abolished by the presence of NGF. alpha9beta1 integrin appears to be an interesting target for blocking the progression of malignant gliomas, especially in light of the stimulatory effect of NGF on the development of these tumors and its ability to transfer proapoptotic signals in cancer cells.

Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site.

The presented results show the effect of targeting of collagen receptor, alpha1beta1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in 2 animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization., ((c) 2008 Wiley-Liss, Inc.)

The integrin alpha9beta1 is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin C and osteopontin. We found that this integrin is a receptor for nerve growth factor (NGF) and two other neurotrophins, brain-derived neurotrophic factor and NT3, using a cell adhesion assay with the alpha9SW480 cell line. Interaction of alpha9beta1 with NGF was confirmed in an ELISA assay by direct binding to purified integrin. alpha9beta1 integrin binds to neurotrophins in a manner similar to another common neurotrophin receptor, p75(NTR) (NGFR), although alpha9beta1 activity is correlated with induction of pro-survival and pro-proliferative signaling cascades. This property of alpha9beta1 resembles the interaction of NGF with a high affinity receptor, TrkA, however, this integrin shows a low affinity for NGF. NGF induces chemotaxis of cells expressing alpha9beta1 and their proliferation. Moreover, alpha9beta1 integrin is a signaling receptor for NGF, which activates the MAPK (Erk1/2) pathway. The alpha9beta1-dependent chemotactic ability of NGF appears to result from the activation of paxillin.

Tumor necrosis factor-alpha (TNFalpha) released in the brain by HIV-activated macrophages/microglia is suspected to compromise neuronal survival. Previously, we have demonstrated that activated receptor for insulin-like growth factor I (IGF-IR) protects neurons from TNFalpha-induced neuronal damage (Wang et al. [ 2006] J. Neurosci. Res. 83:7-18). Because TNFalpha triggers phosphorylation of insulin receptor substrate 1 (IRS-1) on serine residues (pS-IRS-1; Rui et al. [ 2001] J. Clin. Invest. 107:181-189), and pS-IRS-1 binds integrins (Reiss et al. [ 2001] Oncogene 20:490-500), we asked how these events affect neuronal processes. We show that beta1-integrin and pS-IRS-1 colocalize in PC12 cells and in primary cortical neurons. TNFalpha treatment elevated membrane-associated pS-IRS-1, enhanced pS-IRS-1 interaction with beta1-integrin, and attenuated cell attachment to collagen IV. In contrast, IGF-I inhibited pS-IRS-1-beta1-integrin complexes and improved cell attachment. The domain of IRS-1 involved in beta1-integrin binding mapped between amino acids 426 and 740, and the expression of 426-740/IRS-1 mutant attenuated neuronal outgrowth. Our results indicate that TNFalpha facilitates the interaction of pS-IRS-1 and beta1-integrin and destabilizes neuronal processes. IGF-I counteracts TNFalpha-mediated accumulation of pS-IRS-1-beta1-integrin complexes supporting the stability of neuronal processes., (Copyright 2007 Wiley-Liss, Inc.)

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Therapeutic angiogenesis is one of the major approaches in designing new therapies for cardiovascular diseases. vpVEGF was purified from Vipera palestinae venom using two steps of reverse-phase HPLC. Structurally, vpVEGF belongs to the VEGF-F1 family of snake venom proteins, and potently stimulated dHMVEC proliferation in a VEGFR-2 dependent manner. This growth factor appeared to be a chemoattractant for migration of these cells and stimulated their radial migration in a collagen gel. The stimulatory effect on dHMVEC was correlated with activation of the MAPK Erk1/2 signaling pathway. In vivo vpVEGF induced angiogenesis in a Japanese quail assay and in a Matrigel plug assay in mice. Although in the quail assay vpVEGF showed lower activity than hrVEGF-A165 in mammalian-related systems there were no significant differences. The experiments with dHMVEC, as well as angiogenesis in vivo suggest that the pro-angiogenic effect of vpVEGF is related to its interaction with VEGFR-2 (flk-1).

Angiogenesis, the formation of new blood vessels, is tightly regulated by growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The authors hypothesize that nerve growth factor (NGF), a well known neurotrophin, may play a direct angiogenic role. To test this hypothesis, the authors measured the effects of NGF on the natural vascularization of the quail chorioallantoic membrane (CAM). The angiogenic effect of NGF was compared to that of human recombinant VEGF165 (rhVEGF) and basic FGF (rhbFGF). In comparison to phosphate-buffered saline-treated controls, NGFs from different biological sources (mouse, viper, and cobra) increased the rate of angiogenesis in a dose-dependent fashion from 0.5 to 5 microg. For quantitative morphometry, grayscale images of the blood vessels end points of the CAM arteries were binarized for visualization and skeletonized for quantization by fractal analysis. In mouse NGF-treated embryos the fractal dimension (Df), indicative of arterial vessel length and density, increased to 1.266 +/- 0.021 compared to 1.131 +/- 0.018 (p < .001) for control embryos. This effect was similar to that of 0.5 microg rhVEGF (1.290 +/- 0.021, p < .001) and 1.5 microg rhbFGF (1.264 +/- 0.028, p < .001). The mouse NGF-induced angiogenic effect was blocked by 1 microM K252a (1.149 +/- 0.018, p < .001), an antagonist of the NGF/trkA receptor, but not by 1 microM SU-5416 (1.263 +/- 0.029, p < .001), the VEGF/Flk1 receptor antagonist, indicating a direct, selective angiogenic effect of NGF via quail embryo trkA receptor activation. These results confirm previous observations that NGF has angiogenic activity and suggest that this neurotrophin may also play an important role in the cardiovascular system, besides its well-known effects in the nervous system. The angiogenic properties of NGF may be beneficial in engineering new blood vessels and for developing novel antiangiogenesis therapies for cancer.

Insulin-like growth factor 1 receptor (IGF-1R) supports neuronal survival against a wide variety of insults. This includes tumor necrosis factor-alpha (TNFalpha)-mediated neuronal damage, which represents one of the factors suspected to play a role in HIV-associated dementia (HAD). PC12 neurons engineered to express human IGF-1R (PC12/IGF-1R) maintain neuronal processes on collagen IV for several weeks. However, prolonged treatment with TNFalpha caused degeneration of neuronal processes, with no apparent signs of apoptosis. In this process, TNFalpha did not affect IGF-1-mediated phosphorylation of IRS-1, IRS-2, Akt, or Erks. In addition, PC12/IGF-1R cells were found to express predominantly alpha1beta1 integrin, which has high affinity to collagen IV. The treatment of PC12/IGF-1R neurons with a specific alpha1beta1 integrin inhibitor, obtustatin, also caused loss of neuronal processes, accompanied by a quick cell detachment and extensive apoptosis. In the presence of IGF-1, both TNFalpha-induced and obtustatin-induced degeneration of neuronal processes were effectively inhibited. Furthermore, TNFalpha-mediated neuronal degeneration correlated with decreased attachment of PC12/IGF-1R cells to collagen IV and with a reduced level of alpha1beta1 integrin, consistent with a role for this surface protein in the maintenance of neuronal processes. Thus the neuroprotective effects of IGF-1 are not restricted to its antiapoptotic properties but also involve an additional neuroprotective mechanism, by which IGF-1 counteracts the negative effect of TNFalpha on alpha1beta1 integrin-mediated attachment to collagen IV.

In order to check the possibility of producing secondary metabolites, in vitro cultures of A. visnaga callus were established. The best growth of A. visnaga callus was obtained on Murashige and Skoog medium (MS) containing 6-benzyladenine (BA) and alpha-naphtaleneacetic acid (NAA). The study was concentrated on the induction of production of secondary metabolites by exposing callus to abiotic elicitors: benzo(1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester (BION) and a suspension of silica (SiO2) and biotic elicitors: autoclaved lysates of Enterobacter sakazaki and scleroglucan. GC analysis indicated that not-elicited callus of A. visnaga grown in darkness accumulated 2 times more visnagin than the one which was grown under a 16-h photoperiod. The highest accumulation of visnagin was observed in the callus culture elicited with scleroglucan or BION. Scleroglucan induced also the accumulation of khellin in A. visnaga callus. The presented work shows that biosynthesis of pharmacologically important secondary metabolites in A. visnaga cultures could be stimulated by application of elicitors.