Sulfide oxidation promotes hypoxic angiogenesis and neovascularization.

Angiogenic programming in the vascular endothelium is a tightly regulated process for maintaining tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Here, we report that hypoxic upregulation of ·NO in endothelial cells reprograms the transsulfuration pathway to increase biogenesis of hydrogen sulfide (H2S), a proangiogenic metabolite. However, decreased H2S oxidation due to sulfide quinone oxidoreductase (SQOR) deficiency synergizes with hypoxia, inducing a reductive shift and limiting endothelial proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body (WBCreSqorfl/fl) and endothelial-specific (VE-cadherinCre-ERT2Sqorfl/fl) Sqor-knockout mice exhibit lower mass and angiogenesis than control mice. WBCreSqorfl/fl mice also exhibit decreased muscle angiogenesis following femoral artery ligation compared to control mice. Collectively, our data reveal the molecular intersections between H2S, O2 and ·NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.

PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Disruption of hypoxia-inducible factor-2α in neutrophils decreases colitis-associated colon cancer.

Neutrophils are abundant immune cells in the colon tumor microenvironment. Studies have shown that neutrophils are recruited into hypoxic foci in colon cancer. However, the impact of hypoxia signaling on neutrophil function and its involvement in colon tumorigenesis remain unclear. To address this, we generated mice with a deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α in neutrophils driven by the MRP8Cre (HIF-1αΔNeu) or (HIF-2αΔNeu) and littermate controls. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colon cancer, the disruption of neutrophils-HIF-1α did not result in any significant changes in body weight, colon length, tumor size, proliferation, or burden. However, the disruption of HIF-2α in neutrophils led to a slight increase in body weight, a significant decrease in the number of tumors, and a reduction in tumor size and volume compared with their littermate controls. Histological analysis of colon tissue from mice with HIF-2α-deficient neutrophils revealed notable reductions in proliferation as compared with control mice. In addition, we observed reduced levels of proinflammatory cytokines, such as TNF-α and IL-1ß, in neutrophil-specific HIF-2α-deficient mice in both the tumor tissue as well as the neutrophils. Importantly, it is worth noting that the reduced tumorigenesis associated with HIF-2α deficiency in neutrophils was not evident in already established syngeneic tumors or a DSS-induced inflammation model, indicating a potential role of HIF-2α specifically in colon tumorigenesis. In conclusion, we found that the loss of neutrophil-specific HIF-2α slows colon tumor growth and progression by reducing the levels of inflammatory mediators.NEW & NOTEWORTHY Despite the importance of hypoxia and neutrophils in colorectal cancer (CRC), the contribution of neutrophil-specific HIFs to colon tumorigenesis is not known. We describe that neutrophil HIF-1α has no impact on colon cancer, whereas neutrophil HIF-2α loss reduces CRC growth by decreasing proinflammatory and immunosuppressive cytokines. Furthermore, neutrophil HIF-2α does not reduce preestablished tumor growth or inflammation-induced colitis. The present study offers novel potential of neutrophil HIF-2α as a therapeutic target in CRC.

Oxygen battle in the gut: Hypoxia and hypoxia-inducible factors in metabolic and inflammatory responses in the intestine.

The gastrointestinal tract is a highly proliferative and regenerative tissue. The intestine also harbors a large and diverse microbial population collectively called the gut microbiome (microbiota). The microbiome-intestine cross-talk includes a dynamic exchange of gaseous signaling mediators generated by bacterial and intestinal metabolisms. Moreover, the microbiome initiates and maintains the hypoxic environment of the intestine that is critical for nutrient absorption, intestinal barrier function, and innate and adaptive immune responses in the mucosal cells of the intestine. The response to hypoxia is mediated by hypoxia-inducible factors (HIFs). In hypoxic conditions, the HIF activation regulates the expression of a cohort of genes that promote adaptation to hypoxia. Physiologically, HIF-dependent genes contribute to the aforementioned maintenance of epithelial barrier function, nutrient absorption, and immune regulation. However, chronic HIF activation exacerbates disease conditions, leading to intestinal injury, inflammation, and colorectal cancer. In this review, we aim to outline the major roles of physiological and pathological hypoxic conditions in the maintenance of intestinal homeostasis and in the onset and progression of disease with a major focus on understanding the complex pathophysiology of the intestine.

Engulfment of Hb-activated platelets differentiates monocytes into pro-inflammatory macrophages in PNH patients.

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)-activated platelets, classical monocytes differentiated into pro-inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb-activated platelets, monocytes differentiate into M1-macrophages under M1-polarization stimulus (GM-CSF, IFN-γ + LPS). When grown under M2-polarization stimulus (M-CSF, IL-4 + IL13), the cells exhibited an M1-like phenotype, secreted elevated levels of pro-inflammatory cytokines including TNF-α and IL-1ß, and displayed loss of the secretion of cytokine such as IL-10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho-STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte-platelet aggregates in circulation and accumulation of CD80high macrophages in thioglycollate-induced inflamed peritoneum. The spleen of the mice was also populated by CD80high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb-activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.

Unique case of autoantibody mediated inactivation of ADAMTS13 in an Indian TTP patient.

A young Indian female visited hospital as a suspected case of thrombotic thrombocytopenic purpura (TTP) with relapsed thrombotic complications with low platelet counts, infarct in middle cerebral artery and thrombi in microvessels. We first confirmed the deficiency of ADAMTS13 metalloprotease in this patient showing improper cleavage of vWF multimers by her plasma unlike her parents and brother. Although patient had very less ADAMTS13 antigen in plasma, but it did not appear to be the cause of deficiency of the enzyme, because her father had similarly low antigen level and he never had prothrombotic complications. While investigating the genetic change in ADAMTS13, we observed four homozygous-SNPs (g.420T>C, g.1342C>G, g.1716G>A and g.2280T>C) in exon 5, 12, 15 and 19 respectively in patient and her father unlike the heterozygous form of same SNPs in mother and brother. Further to investigate the cause of ADAMTS13 deficiency, we observed an elevated level of antibody against ADAMTS13 in patient unlike her father and other family members. Our study therefore provides the molecular approach of diagnosis of TTP in this patient and also highlights the use of such techniques in India. More importantly, study provides the clue of alternate treatment such as immunosuppressant therapy to this patient.

Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders.

Monocytes and macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin (Hb) in hemolytic conditions. While investigating the phenotype of monocytes in two hemolytic disorders-paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory (CD14+CD16hi) monocytes in these patients. We further investigated in vitro the phenotype of these monocytes and found an estimated 55% of CD14+ cells were transformed into the CD14+CD16hi subset after engulfing Hb-activated platelets. The CD14+CD16hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1ß, unlike monocytes treated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytes engulfed these activated platelets in vivo. As expected, we found 95% of CD14+CD16hi monocytes with both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocytes whether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14+CD16hi monocytes displayed significantly decreased phagocytosis of E. coli. Our study therefore suggests that this alteration of monocyte phenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD.

Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: a novel mechanism associated with intravascular hemolysis.

Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37-3 µM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3-6 µM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation.

Myeloid Hif2α is not essential to maintain systemic iron homeostasis.

Dietary consumption serves as the primary source of iron uptake, and erythropoiesis acts as a major regulator of systemic iron demand. In addition to intestinal iron absorption, macrophages play a crucial role in recycling iron from senescent red blood cells. The kidneys are responsible for the production of erythropoietin (Epo), which stimulates erythropoiesis, whereas the liver plays a central role in producing the iron-regulatory hormone hepcidin. The transcriptional regulator hypoxia-inducible factor (HIF)2α has a central role in the regulation of Epo, hepcidin, and intestinal iron absorption and therefore plays a crucial role in coordinating the tissue crosstalk to maintain systemic iron demands. However, the precise involvement of Hif2α in macrophages in terms of iron homeostasis remains uncertain. Our study demonstrates that deleting Hif2α in macrophages does not disrupt the expression of iron transporters or basal erythropoiesis. Mice lacking Hif2α in myeloid cells exhibited no discernible differences in hemodynamic parameters, including hemoglobin concentrations and erythrocyte count, when compared with littermate controls. This similarity was observed under conditions of both dietary iron deficiency and acute erythropoietic demand. Notably, we observed a significant increase in the expression of iron transporters in the duodenum during iron deficiency, indicating heightened iron absorption. Therefore, our findings suggest that the disruption of Hif2α in myeloid cells does not significantly impact systemic iron homeostasis under normal physiologic conditions. However, its disruption induces adaptive physiologic changes in response to elevated iron demand, potentially serving as a mechanism to sustain increased erythropoietic demand.

Sulfide oxidation promotes hypoxic angiogenesis and neovascularization.

Angiogenic programming in the vascular endothelium is a tightly regulated process to maintain tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Herein, we report that hypoxic upregulation of NO synthesis in endothelial cells reprograms the transsulfuration pathway and increases H 2 S biogenesis. Furthermore, H 2 S oxidation by mitochondrial sulfide quinone oxidoreductase (SQOR) rather than downstream persulfides, synergizes with hypoxia to induce a reductive shift, limiting endothelial cell proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body WB Cre SQOR fl/fl knockout mice exhibit lower mass and reduced angiogenesis compared to SQOR fl/fl controls. WB Cre SQOR fl/fl mice also exhibit reduced muscle angiogenesis following femoral artery ligation, compared to controls. Collectively, our data reveal the molecular intersections between H 2 S, O 2 and NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization. Highlights: Hypoxic induction of •NO in endothelial cells inhibits CBS and switches CTH reaction specificity Hypoxic interruption of the canonical transsulfuration pathway promotes H 2 S synthesis Synergizing with hypoxia, SQOR deficiency induces a reductive shift in the ETC and restricts proliferationSQOR KO mice exhibit lower neovascularization in tumor xenograft and hind limb ischemia models.