VEGF Pathways in the Lymphatics of Healthy and Diseased Heart

Cardiac lymphatic system is a rare focus of the modern cardiovascular research. Nevertheless, the growing body of evidence is depicting lymphatic endothelium as an important functional unit in healthy and diseased myocardium. Since the discovery of angiogenic VEGF‐A in 1983 and lymphangiogenic VEGF‐C in 1997, an increasing amount of knowledge has accumulated on the essential roles of VEGF ligands and receptors in physiological and pathological angiogenesis and lymphangiogenesis. Tissue adaptation to several stimuli such as hypoxia, pathogen invasion, degenerative process and inflammation often involves coordinated changes in both blood and lymphatic vessels. As lymphatic vessels are involved in the initiation and resolution of inflammation and regulation of tissue edema, VEGF family members may have important roles in myocardial lymphatics in healthy and in cardiac disease. We will review the properties of VEGF ligands and receptors concentrating on their lymphatic vessel effects first in normal myocardium and then in cardiac disease.     

Ferristatin II Efficiently Inhibits SARS-CoV-2 Replication in Vero Cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC₅₀ values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy.     

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

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

Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette

OBJECTIVE: Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q and R type) coordinate a variety of Ca(2+)-dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type alpha1E (Ca(v)2.3) subunit is expressed as the tissue-specific splice variant alpha1Ee. DESIGN AND METHODS: To understand the functional role of alpha1E-containing Ca(2+) channels, antisense alpha1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense alpha1E cassette cDNA. As controls, either a sense alpha1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR. RESULTS: In three independent antisense alpha1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense alpha1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense alpha1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent. CONCLUSION: alpha1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.     

Single-channel pharmacology of mibefradil in human native T-type and recombinant Ca(v)3.2 calcium channels

To study the molecular pharmacology of low-voltage-activated calcium channels in biophysical detail, human medullary thyroid carcinoma (hMTC) cells were investigated using the single-channel technique. These cells had been reported to express T-type whole-cell currents and a Ca(v)3.2 (or alpha 1H) channel subunit. We observed two types of single-channel activity that were easily distinguished based on single-channel conductance, voltage dependence of activation, time course of inactivation, rapid gating kinetics, and the response to the calcium agonist (S)-Bay K 8644. Type II channels had biophysical properties (activation, inactivation, conductance) typical for high-voltage-activated calcium channels. They were markedly stimulated by 1 microM (S)-Bay K 8644, allowing to identify them as L-type channels. The channel termed type I is a low-voltage-activated, small-conductance (7.2 pS) channel that inactivates rapidly and is not modulated by (S)-Bay K 8644. Type I channels are therefore classified as T-type channels. They were strongly inhibited by 10 microM mibefradil. Mibefradil block was caused by changes in two gating parameters: a pronounced reduction in fraction of active sweeps and a slight shortening of the open-state duration. Single recombinant low-voltage-activated T-type calcium channels were studied in comparison, using human embryonic kidney 293 cells overexpressing the pore-forming Ca(v)3.2 subunit. Along all criteria examined (mechanisms of block, extent of block), recombinant Ca(v)3.2 interact with mibefradil in the same way as their native counterparts expressed in hMTC cells. In conclusion, the pharmacologic phenotype of these native human T-type channels--as probed by mibefradil--is similar to recombinant human Ca(v)3.2.     

Relative picrotoxin insensitivity distinguishes ionotropic GABA receptor-mediated IPSCs in hippocampal interneurons

Inhibitory GABAergic signalling in the hippocampus plays an important role in synchronizing principal cells and regulating the excitability of this seizure-prone structure. Distinct mechanisms modulate release from GABAergic terminals in the hippocampus, depending on whether the postsynaptic partner is an interneuron or a principal cell. Here, we report that postsynaptic ionotropic GABA receptors in principal cells and interneurons also show a striking pharmacological difference. The broad-spectrum antagonist picrotoxin (PTX) was less potent at blocking IPSCs evoked in stratum radiatum interneurons than in pyramidal neurons in the CA1 region. GABA-evoked currents in membrane patches from interneurons showed a smaller mean unitary conductance than in patches from pyramidal neurons. Because retinal GABA(C) receptors show decreased picrotoxin sensitivity and conductance, we examined the effect of the GABA(C) receptor agonist cis-aminocrotonic acid (CACA). Although this agent evoked picrotoxin-resistant currents in interneurons, these were enhanced by the GABA(A) allosteric modulator pentobarbital. Moreover, both picrotoxin-resistant IPSCs and CACA-evoked currents were blocked by the GABA(A) receptor-selective antagonist bicuculline. The presence of relatively picrotoxin-resistant GABA(A) receptors in interneurons provides a potential target for agents to modulate the activity of sub-populations of hippocampal neurons.     

Respiratory insufficiency in desminopathy patients caused by introduction of proline residues in desmin c-terminal alpha-helical segment

Mutations in desmin gene have been identified in patients with cardiac and skeletal myopathy characterized by intracytoplasmic accumulation of desmin-reactive deposits and electron-dense granular aggregates. We characterized two new desminopathy families with unusual features of adult-onset, slowly progressive, diffuse skeletal myopathy and respiratory insufficiency. Progressive reduction of respiratory muscle strength became clinically detectable between the 3rd and the 8th years of illness and led to recurrent chest infections and death in one of the patients. Novel mutations, A357P and L370P, predicted to introduce proline residue into a highly conserved alpha-helical region of desmin, were identified. Proline is known to disrupt the alpha-helix. In addition, the A357P mutation distorts a unique stutter sequence that is considered to be critically important for proper filament assembly. Functional assessment in two cell-lines, one of which does and the other of which does not constitutively produce type III intermediate filaments, demonstrated the inability of mutant desmin carrying either the A357P or the L370P mutation to polymerize and form an intracellular filamentous network. The results of this study indicate that respiratory insufficiency is an intrinsic feature of disease associated with specific desmin mutations; in some patients, respiratory weakness may present as a dominant clinical manifestation and a major cause of disability and death.