Mitochondrial dysfunction induced by callyspongiolide promotes autophagy-dependent cell death.

Callyspongiolide is a marine macrolide known to induce caspaseindependent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments. [BMB Reports 2021; 54(4): 227-232].

MicroRNA-148a/b-3p regulates angiogenesis by targeting neuropilin-1 in endothelial cells.

MicroRNAs (miRs) are crucial regulators of vascular endothelial cell (EC) functions, including migration, proliferation, and survival. However, the role of most miRs in ECs remains unknown. Using RNA sequencing analysis, we found that miR-148a/b-3p expression was significantly downregulated during the differentiation of umbilical cord blood mononuclear cells into outgrowing ECs and that decreased miR-148a/b-3p levels were closely related to EC behavior. Overexpression of miR-148a/b-3p in ECs significantly reduced migration, filamentous actin remodeling, and angiogenic sprouting. Intriguingly, the effects of decreased miR-148a/b-3p levels were augmented by treatment with vascular endothelial growth factor (VEGF). Importantly, we found that miR-148a/b-3p directly regulated neuropilin-1 (NRP1) expression by binding to its 3'-untranslated region. In addition, because NRP1 is the coreceptor for VEGF receptor 2 (VEGFR2), overexpression of miR-148a/b-3p inhibited VEGF-induced activation of VEGFR2 and inhibited its downstream pathways, as indicated by changes to phosphorylated focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase. Collectively, our results demonstrate that miR-148a/b-3p is a direct transcriptional regulator of NRP1 that mediates antiangiogenic pathways. These data suggest that miR-148a/b-3p is a therapeutic candidate for overcoming EC dysfunction and angiogenic disorders, including ischemia, retinopathy, and tumor vascularization.

Efficacy and tolerability of desensitization in the treatment of delayed drug hypersensitivities to anti-tuberculosis medications.

BACKGROUND: Delayed drug hypersensitivity to first-line anti-tuberculosis medication is a major challenge in tuberculosis treatment. OBJECTIVE: This study was performed to investigate the efficacy/tolerability of desensitization therapy in treatment of first-line anti-tuberculosis medication hypersensitivity and the usefulness of immunologic evaluation therein. METHODS: This study was conducted as a prospective, observational cohort study. Subjects who experienced hypersensitivity reactions, including maculopapular exanthema (MPE) and drug reaction with eosinophilia and systemic symptoms (DRESS), to first-line anti-tuberculosis medications (isoniazid [INH], ethambutol [EMB], rifampin [RFP], and pyrazinamide [PZA]) were enrolled. Patch, intradermal, lymphocyte transformation, and oral provocation tests were performed to determine culprit drugs, which were desensitized with rapid and graded challenge protocols. Breakthrough reactions (BTRs) during or after desensitization were assessed. RESULTS: In total, 31 desensitization treatments (INH, 8; EMB, 8; RFP, 11; PZA, 4) to 12 patients (8 with MPE and 4 with DRESS) were performed. The overall success rate of desensitization was 80.7%. All the study subjects except one completed the full course of anti-tuberculosis treatment. The overall BTR free rate was 64.5%. Sixteen (80%) treatments for MPE and four (36.4%) for DRESS were BTR free (P = 0.023). Drugs that were positive on any two of three immunologic studies showed significantly high BTR rates (P = 0.014), although this was not correlated with desensitization failure rate. CONCLUSION: Rapid desensitization therapy to multiple anti-tuberculosis medications for delayed drug hypersensitivity was safe and successful. Combination of multiple immunologic evaluations may predict BTR although it needs validation in larger studies.

Integrative analysis of DNA methylation and mRNA expression during differentiation of umbilical cord blood derived mononuclear cells to endothelial cells.

Differentiation of umbilical cord blood derived mononuclear cells to endothelial cells is accompanied by massive changes in gene expression. Although methylation and demethylation of DNA likely play crucial roles in regulating gene expression, their interplay during differentiation remains elusive. To address this question, we performed deep sequencing of DNA methylation and mRNA expression to profile global changes in promoter methylation and gene expression during differentiation from mononuclear cells to outgrowing cells. We identified 61 downregulated genes with hypermethylation, including CD74, VAV1, TLR8, and NCF4, as well as 21 upregulated genes with hypomethylation, including ECSCR, MCAM, PGF, and ARHGEF15. Interestingly, gene ontology analysis showed that downregulated genes with hypermethylation were enriched in immune-related functions, and upregulated genes with hypomethylation were enriched in the developmental process and angiogenesis, indicating the important roles of DNA methylation in regulating differentiation. We performed polymerase chain reaction analyses and bisulfite sequencing of representative genes (CD74, VAV1, ECSCR, and MCAM) to verify the negative correlation between DNA methylation and gene expression. Further, inhibition of DNA methyltransferase and demethylase activities using 5'-aza-dc and shRNAs, specific for TET1 and TET2 mRNAs, respectively, revealed that DNA methylation was the main regulator of the reversible expression of functionally important genes. Collectively, our findings implicate DNA methylation as a critical regulator of gene expression during umbilical cord blood derived mononuclear cells to endothelial cell differentiation.

Sac-1004, a vascular leakage blocker, reduces cerebral ischemia-reperfusion injury by suppressing blood-brain barrier disruption and inflammation.

BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. RESULTS: Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1ß-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. CONCLUSIONS: Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.

The endothelial E3 ligase HECW2 promotes endothelial cell junctions by increasing AMOTL1 protein stability via K63-linked ubiquitination.

Cell-to-cell junctions are critical for the formation of endothelial barriers, and its disorganization is required for sprouting angiogenesis. Members of the angiomotin (AMOT) family have emerged as key regulators in the control of endothelial cell (EC) junction stability and permeability. However, the underlying mechanism by which the AMOT family is regulated in ECs remains unclear. Here we report that HECW2, a novel EC ubiquitin E3 ligase, plays a critical role in stabilizing endothelial cell-to-cell junctions by regulating AMOT-like 1 (AMOTL1) stability. HECW2 physically interacts with AMOTL1 and enhances its stability via lysine 63-linked ubiquitination. HECW2 depletion in human ECs decreases AMOTL1 stability, loosening the cell-to-cell junctions and altering subcellular localization of yes-associated protein (YAP) from cytoplasm into the nucleus. Knockdown of HECW2 also results in increased angiogenic sprouting, and this effect is blocked by depletion of ANG-2, a potential target of YAP. These results demonstrate that HECW2 is a novel regulator of angiogenesis and provide new insights into the mechanisms coordinating junction stability and angiogenic activation in ECs.

BMP9 Induces Cord Blood-Derived Endothelial Progenitor Cell Differentiation and Ischemic Neovascularization via ALK1.

OBJECTIVE: Modulating endothelial progenitor cells (EPCs) is essential for therapeutic angiogenesis, and thus various clinical trials involving EPCs are ongoing. However, the identification of environmental conditions and development of optimal methods are required to accelerate EPC-driven vasculogenesis. APPROACH AND RESULTS: We evaluated gene expression profiles of cord blood-derived EPCs and endothelial cells to identify the key factors in EPC→endothelial cell differentiation and to show that transforming growth factor-ß family members contribute to EPC differentiation. The expression levels of activin receptor-like kinase 1 (ALK1) and its high-affinity ligand, bone morphogenetic protein 9 (BMP9) were markedly changed in EPC→endothelial cell differentiation. Interestingly, BMP9 induced EPC→endothelial cell differentiation and EPC incorporation into vessel-like structures by acting on ALK1 expressed on EPCs in vitro. BMP9 also induced neovascularization in mice with hindlimb ischemia by increasing vessel formation and the incorporation of EPCs into vessels. Conversely, neovascularization was impaired when ALK1 signaling was blocked. Furthermore, EPCs exposed to either short- or long-term BMP9 stimulation demonstrated these functions in EPC-mediated neovascularization. CONCLUSIONS: Collectively, our results indicated that BMP9/ALK1 augmented vasculogenesis and angiogenesis, and thereby enhanced neovascularization. Thus, we suggest that BMP9/ALK1 may improve the efficacy of EPC-based therapies for treating ischemic diseases.