Epigenetic targeting of the ACE2 and NRP1 viral receptors limits SARS-CoV-2 infectivity.

BACKGROUND: SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors for entry into cells, and the serine protease TMPRSS2 for S protein priming. Inhibition of protease activity or the engagement with ACE2 and NRP1 receptors has been shown to be an effective strategy for blocking infectivity and viral spreading. Valproic acid (VPA; 2-propylpentanoic acid) is an epigenetic drug approved for clinical use. It produces potent antiviral and anti-inflammatory effects through its function as a histone deacetylase (HDAC) inhibitor. Here, we propose VPA as a potential candidate to tackle COVID-19, in which rapid viral spread and replication, and hyperinflammation are crucial elements. RESULTS: We used diverse cell lines (HK-2, Huh-7, HUVEC, Caco-2, and BEAS-2B) to analyze the effect of VPA and other HDAC inhibitors on the expression of the ACE-2 and NRP-1 receptors and their ability to inhibit infectivity, viral production, and the inflammatory response. Treatment with VPA significantly reduced expression of the ACE2 and NRP1 host proteins in all cell lines through a mechanism mediated by its HDAC inhibitory activity. The effect is maintained after SARS-CoV-2 infection. Consequently, the treatment of cells with VPA before infection impairs production of SARS-CoV-2 infectious viruses, but not that of other ACE2- and NRP1-independent viruses (VSV and HCoV-229E). Moreover, the addition of VPA 1 h post-infection with SARS-CoV-2 reduces the production of infectious viruses in a dose-dependent manner without significantly modifying the genomic and subgenomic messenger RNAs (gRNA and sg mRNAs) or protein levels of N protein. The production of inflammatory cytokines (TNF-α and IL-6) induced by TNF-α and SARS-CoV-2 infection is diminished in the presence of VPA. CONCLUSIONS: Our data showed that VPA blocks three essential processes determining the severity of COVID-19. It downregulates the expression of ACE2 and NRP1, reducing the infectivity of SARS-CoV-2; it decreases viral yields, probably because it affects virus budding or virions stability; and it dampens the triggered inflammatory response. Thus, administering VPA could be considered a safe treatment for COVID-19 patients until vaccines have been rolled out across the world.

Regulatory T cells as targets for immunotherapy of autoimmunity and inflammation.

Regulatory T (Treg) cells are emerging as key players in the regulation of different immune responses, thereby representing potential candidates for therapeutic interventions in a broad variety of immunological disorders. While the reduction or loss in function would be of benefit during the treatment of cancer, induction and/or expansion of Treg cell function might be helpful to interfere with unwanted immune responses in transplantation medicine, during autoimmunity, allergy and inflammation. However, a better understanding of Treg cell biology is a prerequisite to specifically modulate its function during immune responses in vivo. In the present review we will discuss current concepts on different cell types, components and some novel surface receptors expressed by Treg cells, namely Neuropilin-1, CD83 and G protein-coupled receptor 83 which might represent promising targets for the modulation of Treg cell function in human disease.

Targeting angiogenesis driven by vascular endothelial growth factors using antibody-based therapies.

PURPOSE: Angiogenesis, one of the hallmarks of cancer, has recently become the target of therapeutic approaches in oncology. Among the complex system of pro- and antiangiogenic factors, the vascular endothelial growth factor system stands out as key mediator of tumor-initiated angiogenesis and as target of antiangiogenesis agents introduced in clinical practice. Although antivascular endothelial growth factor therapies, and in particular, bevacizumab as monoclonal antibody against vascular endothelial growth factor has clearly demonstrated antitumor efficacy, its mechanism of action is not fully understood. DESIGN: This review will discuss the rationale for using antiangiogenesis as anticancer therapy with focus on antibody-based approaches toward the vascular endothelial growth factor-system. Results of clinical trials using bevacizumab will be discussed in detail. RESULTS: Bevacizumab has well-documented efficacy as part of first-line therapy in various malignancies ranging from colorectal to breast and lung cancer. Although it mainly exerts its efficacy in conjunction with conventional cytotoxic chemotherapy, several, apparently vascular endothelial growth factor-dependent malignancies such as renal cell cancer, ovarian cancer, and glioblastoma have shown to be susceptible to single-agent bevacizumab. DISCUSSION: Antiangiogenesis therapy with antibodies, namely bevacizumab as antivascular endothelial growth factor agent, has demonstrated efficacy in various human malignancies. The mechanism of action of antivascular endothelial growth factor therapy in general and bevacizumab in particular, however, is not fully understood. Predictive markers have not yet been identified and questions regarding bevacizumab's usefulness in the adjuvant setting as well as its value as continued therapy beyond progression are still unanswered. It is indisputable, though, that antiangiogenesis has greatly enhanced the therapeutic arsenal of anticancer therapies and has changed oncology forever.

Neuropilin-1 in acute myeloid leukemia: expression and role in proliferation and migration of leukemia cells.

Neuropilin-1 (NRP-1) is a novel receptor of vascular endothelial growth factor (VEGF) and expressed in endothelial cells and tumor cells. The role of NRP-1 in the growth and progression of leukemia is unknown. Here we studied the mRNA expression and effect of NRP-1 in leukemic cells. Our results showed that NRP-1 mRNA was expressed in six of seven leukemic cell lines and primary leukemias derived from all 24 patients with acute myeloid leukemia (AML). Reduced NRP-1 expression by RNA interference led to a decrease of VEGF-mediated mitogenic and migration responses in acute myeloid leukemic cell line HEL. Increased NRP-1 expression was directly correlated with the blast percentage in both peripheral blood and bone marrow of AML patients. Our data demonstrated that a higher level of NRP-1 mRNA was expressed in leukemias and NRP-1 promoted proliferation and chemotaxis of leukemic cells in response to VEGF. Inhibition of NRP-1 functions may provide a new therapeutic strategy for treatment of AML.

Downregulation of neuropilin-1 in patients with acute myeloid leukemia treated with thalidomide.

OBJECTIVE: Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor functioning as a mediator of angiogenesis and neuronal guidance, was recently found to be significantly overexpressed in newly diagnosed acute myeloid leukemia (AML) patients with significant correlation to survival. The role of NRP-1 in refractory or relapsed AML patients and its regulation during anti-angiogenic treatment remain to be elucidated. METHODS: Bone marrow biopsies of 10 patients with refractory or relapsed AML were evaluated for NRP-1 expression by immunohistochemical analysis, and NRP-1 expression level was determined before and after start of thalidomide therapy and correlated to response and growth factor expression. RESULTS: NRP-1 expression was significantly increased in AML patients [median 7 arbitrary units (AU)] when compared with controls (n = 38, median 2.75 AU). Under thalidomide treatment, a marked difference in the course of NRP-1 expression between responders and non-responders was observed, however, without a statistical significance (P = 0.071) being reached. Additionally, a significant correlation of the NRP-1 expression level to microvessel density could be detected under treatment with thalidomide (P = 0.018). CONCLUSION: Our data provide evidence of increased NRP-1 expression in relapsed or refractory AML. Additionally, our results suggest that thalidomide-induced antileukemic properties might at least in part be mediated by NRP-1 downregulation.

Relative effects of VEGF-A and VEGF-C on endothelial cell proliferation, migration and PAF synthesis: Role of neuropilin-1.

Vascular endothelial growth factor (VEGF-A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet-activating factor (PAF). Recently, neuropilin-1 (NRP-1) has been described as a coreceptor of KDR which potentiates VEGF-A activity. However, the role of NRP-1 in numerous VEGF-A activities remains unclear. To assess the contribution of NRP-1 to VEGF-A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt-1, NRP-1, KDR or KDR and NRP-1. Cells were stimulated with VEGF-A, which binds to Flt-1, KDR and NRP-1, and VEGF-C, which binds to KDR only. VEGF-A was 12.4-fold more potent than VEGF-C in inducing KDR phosphorylation in PAEC-KDR. VEGF-A and VEGF-C showed similar potency to mediate PAEC-KDR proliferation, migration, and PAF synthesis. On PAEC-KDR/NRP-1, VEGF-A was 28.6-fold more potent than VEGF-C in inducing KDR phosphorylation and PAEC-KDR/NRP-1 proliferation (1.3-fold), migration (1.7-fold), and PAF synthesis (4.6-fold). These results suggest that cooperative binding of VEGF-A to KDR and NRP-1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP-1 receptors. In contrast, stimulation of PAEC-Flt-1 and PAEC-NRP-1 with VEGF-A or VEGF-C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP-1 on EC preferentially increases KDR activation by VEGF-A as well as KDR-mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF-A and VEGF-C have equipotent biological activities on EC in absence of NRP-1.