Targeting branched N-glycans and fucosylation sensitizes ovarian tumors to immune checkpoint blockade.

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it's unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8+ T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N-glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion.

Intraperitoneal metastasis of ovarian cancer: new insights on resident macrophages in the peritoneal cavity.

Ovarian cancer metastasis occurs primarily in the peritoneal cavity. Orchestration of cancer cells with various cell types, particularly macrophages, in the peritoneal cavity creates a metastasis-favorable environment. In the past decade, macrophage heterogeneities in different organs as well as their diverse roles in tumor settings have been an emerging field. This review highlights the unique microenvironment of the peritoneal cavity, consisting of the peritoneal fluid, peritoneum, and omentum, as well as their own resident macrophage populations. Contributions of resident macrophages in ovarian cancer metastasis are summarized; potential therapeutic strategies by targeting such cells are discussed. A better understanding of the immunological microenvironment in the peritoneal cavity will provide a stepping-stone to new strategies for developing macrophage-based therapies and is a key step toward the unattainable eradication of intraperitoneal metastasis of ovarian cancer.

Targeting the mevalonate pathway suppresses ARID1A-inactivated cancers by promoting pyroptosis.

ARID1A, encoding a subunit of the SWI/SNF complex, is mutated in ∼50% of clear cell ovarian carcinoma (OCCC) cases. Here we show that inhibition of the mevalonate pathway synergizes with immune checkpoint blockade (ICB) by driving inflammasome-regulated immunomodulating pyroptosis in ARID1A-inactivated OCCCs. SWI/SNF inactivation downregulates the rate-limiting enzymes in the mevalonate pathway such as HMGCR and HMGCS1, which creates a dependence on the residual activity of the pathway in ARID1A-inactivated cells. Inhibitors of the mevalonate pathway such as simvastatin suppresses the growth of ARID1A mutant, but not wild-type, OCCCs. In addition, simvastatin synergizes with anti-PD-L1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation and in a humanized immunocompetent ARID1A mutant patient-derived OCCC mouse model. Our data indicate that inhibition of the mevalonate pathway simultaneously suppresses tumor cell growth and boosts antitumor immunity by promoting pyroptosis, which synergizes with ICB in suppressing ARID1A-mutated cancers.

Oxygen Gradient Induced in Microfluidic Chips Can Be Used as a Model for Liver Zonation.

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production.

Disabling partners in crime: Gold nanoparticles disrupt multicellular communications within the tumor microenvironment to inhibit ovarian tumor aggressiveness.

The tumor microenvironment (TME) plays a key role in the poor prognosis of many cancers. However, there is a knowledge gap concerning how multicellular communication among the critical players within the TME contributes to such poor outcomes. Using epithelial ovarian cancer (EOC) as a model, we show how crosstalk among cancer cells (CC), cancer associated fibroblasts (CAF), and endothelial cells (EC) promotes EOC growth. We demonstrate here that co-culturing CC with CAF and EC promotes CC proliferation, migration, and invasion in vitro and that co-implantation of the three cell types facilitates tumor growth in vivo. We further demonstrate that disruption of this multicellular crosstalk using a gold nanoparticle (GNP) inhibits these pro-tumorigenic phenotypes in vitro as well as tumor growth in vivo. Mechanistically, GNP treatment reduces expression of several tumor-promoting cytokines and growth factors, resulting in inhibition of MAPK and PI3K-AKT activation and epithelial-mesenchymal transition - three key oncogenic signaling pathways responsible for the aggressiveness of EOC. The current work highlights the importance of multicellular crosstalk within the TME and its role for the aggressive nature of EOC, and demonstrates the disruption of these multicellular communications by self-therapeutic GNP, thus providing new avenues to interrogate the crosstalk and identify key perpetrators responsible for poor prognosis of this intractable malignancy.

N-Acetylcysteine Inhibits Aortic Stenosis Progression in a Murine Model by Blocking Shear-Induced Activation of Platelet Latent Transforming Growth Factor Beta 1.

Objective: Aortic stenosis (AS) is characterized by narrowing of the aortic valve opening, resulting in peak blood flow velocity that induces high wall shear stress (WSS) across the valve. Severe AS leads to heart failure and death. There is no treatment available for AS other than valve replacement. Platelet-derived transforming growth factor beta 1 (TGF-ß1) partially contributes to AS progression in mice, and WSS is a potent activator of latent TGF-ß1. N-acetylcysteine (NAC) inhibits WSS-induced TGF-ß1 activation in vitro. We hypothesize that NAC will inhibit AS progression by inhibiting WSS-induced TGF-ß1 activation. Approach: We treated a cohort of Ldlr(-/-)Apob(100/100) low density lipoprotein receptor (LDLR) mice fed a high-fat diet with NAC (2% in drinking water) at different stages of disease progression and measured its effect on AS progression and TGF-ß1 activation. Results: Short-term NAC treatment inhibited AS progression in mice with moderate and severe AS relative to controls, but not in LDLR mice lacking platelet-derived TGF-ß1 (TGF-ß1platlet-KO-LDLR). NAC treatment reduced TGF-ß signaling, p-Smad2 and collagen levels, and mesenchymal transition from isolectin B4 and CD45-positive cells in LDLR mice. Mechanistically, NAC treatment resulted in plasma NAC concentrations ranging from 75.5 to 449.2 ng/mL, which were sufficient to block free thiol labeling of plasma proteins and reduce active TGF-ß1 levels without substantially affecting reactive oxygen species-modified products in valvular cells. Conclusions: Short-term treatment with NAC inhibits AS progression by inhibiting WSS-induced TGF-ß1 activation in the LDLR mouse model of AS, motivating a clinical trial of NAC and/or other thiol-reactive agent(s) as a potential therapy for AS.

Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.

Mutations in the human cystathionine beta synthase (CBS) gene are known to cause endothelial dysfunction responsible for cardiovascular and neurovascular diseases. CBS is the predominant hydrogen sulfide (H2 S)-producing enzyme in endothelial cells (ECs). Recently, H2 S was shown to attenuate ROS and improve mitochondrial function. Mitochondria are metabolic organelles that actively transform their ultrastructure to mediate their function. Therefore, we questioned whether perturbation of CBS/H2 S activity could drive mitochondrial dysfunction via mitochondrial dynamics in ECs. Here we demonstrate that silencing CBS induces mitochondria fragmentation, attenuates efficient oxidative phosphorylation, and decreases EC function. Mechanistically, CBS silencing significantly elevates ROS production, thereby leading to reduced mitofusin 2 (MFN2) expression, decouple endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death. These defects were significantly rescued by the treatment of H2 S donors. Taken together our data highlights a novel signaling axis that mechanistically links CBS with mitochondrial function and ER-mitochondrial tethering and could be considered as a new therapeutic approach for the intervention of EC dysfunction-related pathologies.

Hydrogen sulfide signaling in mitochondria and disease.

Hydrogen sulfide can signal through 3 distinct mechanisms: 1) reduction and/or direct binding of metalloprotein heme centers, 2) serving as a potent antioxidant through reactive oxygen species/reactive nitrogen species scavenging, or 3) post-translational modification of proteins by addition of a thiol (-SH) group onto reactive cysteine residues: a process known as persulfidation. Below toxic levels, hydrogen sulfide promotes mitochondrial biogenesis and function, thereby conferring protection against cellular stress. For these reasons, increases in hydrogen sulfide and hydrogen sulfide-producing enzymes have been implicated in several human disease states. This review will first summarize our current understanding of hydrogen sulfide production and metabolism, as well as its signaling mechanisms; second, this work will detail the known mechanisms of hydrogen sulfide in the mitochondria and the implications of its mitochondrial-specific impacts in several pathologic conditions.-Murphy, B., Bhattacharya, R., Mukherjee, P. Hydrogen sulfide signaling in mitochondria and disease.

Inactivation of platelet-derived TGF-ß1 attenuates aortic stenosis progression in a robust murine model.

Aortic stenosis (AS) is a degenerative heart condition characterized by fibrosis and narrowing of aortic valves (AV), resulting in high wall shear stress (WSS) across valves. AS is associated with high plasma levels of transforming growth factor-ß1 (TGF-ß1), which can be activated by WSS to induce organ fibrosis, but the cellular source of TGF-ß1 is not clear. Here, we show that platelet-derived TGF-ß1 plays an important role in AS progression. We first established an aggressive and robust murine model of AS, using the existing Ldlr -/- Apob100/100 (LDLR) breed of mice, and accelerated AS progression by feeding them a high-fat diet (HFD). We then captured very high resolution images of AV movement and thickness and of blood flow velocity across the AV, using a modified ultrasound imaging technique, which revealed early evidence of AS and distinguished different stages of AS progression. More than 90% of LDLR animals developed AS within 6 months of HFD. Scanning electron microscopy and whole-mount immunostaining imaging of AV identified activated platelets physically attached to valvular endothelial cells (VEC) expressing high phosphorylated Smad2 (p-Smad2). To test the contribution of platelet-derived TGF-ß1 in AS, we derived LDLR mice lacking platelet TGF-ß1 (TGF-ß1platelet-KO-LDLR) and showed reduced AS progression and lower p-Smad2 and myofibroblasts in their AV compared with littermate controls fed the HFD for 6 months. Our data suggest that platelet-derived TGF-ß1 triggers AS progression by inducing signaling in VEC, and their subsequent transformation into collagen-producing-myofibroblasts. Thus, inhibiting platelet-derived TGF-ß1 might attenuate or prevent fibrotic diseases characterized by platelet activation and high WSS, such as AS.

Probing Cellular Processes Using Engineered Nanoparticles.

Nanoparticles, the building blocks of nanotechnology, have been widely utilized in various biomedical applications, such as detection, diagnosis, imaging, and therapy. However, another emerging, albeit under-represented, area is the employment of nanoparticles as tools to understand cellular processes (e.g., oxidative stress-induced signaling cascades). Such investigations have enormous potential to characterize a disease from a different perspective and unravel some new features that otherwise would have remained a mystery. In this review, we summarize the intrinsic biological properties of unmodified as well surface modified nanoparticles and discuss how such properties could be utilized to interrogate biological processes and provide a perspective for future evolution of this field.

Platelet TGF-ß1 deficiency decreases liver fibrosis in a mouse model of liver injury.

Transforming growth factor-ß1 (TGF-ß1) signaling in hepatic stellate cells (HSCs) plays a primary role in liver fibrosis, but the source of TGF-ß1 is unclear. Because platelets are rich in TGF-ß1, we examined the role of platelet TGF-ß1 in liver fibrosis by challenging wild-type (WT) mice and mice deficient in platelet TGF-ß1 (PF4CreTgfb1f/f) with carbon tetrachloride (CCl4), an inducer of acute hepatic injury and chronic fibrosis. CCl4 elicited equivalent hepatic injury in WT and PF4CreTgfb1f/f mice based on loss of cytochrome P450 (Cyp2e1) expression, observed at 6 hours and peaking at 3 days after CCl4 challenge; PF4CreTgfb1f/f mice exhibited less liver fibrosis than control mice. Activated platelets were observed during acute liver injury (6 hours), and WT mice with transient platelet depletion (thrombocytopenia) were partially protected from developing fibrosis compared with control mice (P = .01), suggesting an association between platelet activation and fibrosis. Transient increases in TGF-ß1 levels and Smad2 phosphorylation signaling were observed 6 hours and 3 days, respectively, after CCl4 challenge in WT, but not PF4CreTgfb1f/f , mice, suggesting that increased TGF-ß1 levels originated from platelet-released TGF-ß1 during the initial injury. Numbers of collagen-producing HSCs and myofibroblasts were higher at 3 days and 36 days, respectively, in WT vs PF4CreTgfb1f/f mice, suggesting that platelet TGF-ß1 may have stimulated HSC transdifferentiation into myofibroblasts. Thus, platelet TGF-ß1 partially contributes to liver fibrosis, most likely by initiating profibrotic signaling in HSCs and collagen synthesis. Further studies are required to evaluate whether blocking platelet and TGF-ß1 activation during acute liver injury prevents liver fibrosis.

Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Deregulation of mitochondrial morphogenesis, a dynamic equilibrium between mitochondrial fusion and fission processes, is now evolving as a key metabolic event that fuels tumor growth and therapy resistance. However, fundamental knowledge underpinning how cancer cells reprogram mitochondrial morphogenesis remains incomplete. Here, we report that cystathionine ß-synthase (CBS) reprograms mitochondrial morphogenesis in ovarian cancer (OvCa) cells by selectively regulating the stability of mitofusin 2 (MFN2). Clinically, high expression of both CBS and MFN2 implicates poor overall survival of OvCa patients, and a significant association between CBS and MFN2 expression exists in individual patients in the same data set. The silencing of CBS by small interfering RNA or inhibition of its catalytic activity by a small molecule inhibitor creates oxidative stress that activates JNK. Activated JNK phosphorylates MFN2 to recruit homologous to the E6-AP carboxyl terminus' domain-containing ubiquitin E3 ligase for its degradation via the ubiquitin-proteasome system. Supplementation with hydrogen sulfide or glutathione (the catalytic products of CBS enzymatic activity), anti-oxidants, or a JNK inhibitor restores MFN2 expression. In CBS-silenced orthotopic xenograft tumor tissues, MFN2 but not MFN1 is selectively downregulated. In summary, this report reveals a role for deregulated mitochondrial morphogenesis in OvCa, suggests one of the mechanisms for this deregulation, and provides a way to correct it through modulation of the metabolic enzyme CBS.-Chakraborty, P. K., Murphy, B., Mustafi, S. B., Dey, A., Xiong, X., Rao, G., Naz, S., Zhang, M., Yang, D., Dhanasekaran, D. N., Bhattacharya, R., Mukherjee, P. Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Long- but not short-term estradiol treatment induces renal damage in midlife ovariectomized Long-Evans rats.

Clinical recommendations limit menopausal hormone therapy to a few years, yet the impact of a shorter treatment duration on cardiovascular health is unknown. We hypothesized that both short- and long-term estradiol (E2) treatment exerts positive and lasting effects on blood pressure, vascular reactivity, and renal health. This study was designed to mimic midlife menopause, followed by E2 treatment, that either followed or exceeded the current clinical recommendations. Female Long-Evans retired breeders were ovariectomized (OVX) at 11 mo of age and randomized into three groups: 80-day (80d) vehicle (Veh>Veh), 40-day (40d) E2 + 40d vehicle (E2>Veh), and 80d E2 (E2>E2). In comparison to Veh>Veh, both the E2>Veh and E2>E2 groups had lower systolic blood pressure and enhanced mesenteric relaxation in response to estrogen receptor-α stimulation. Despite the reduced blood pressure, E2>E2 induced renal and cardiac hypertrophy, reduced glomerular filtration, and increased proteinuria. Interestingly, kidneys from E2>Veh rats had significantly fewer tubular casts than both of the other groups. In conclusion, long-term E2 lowered blood pressure but exerted detrimental effects on kidney health in midlife OVX Long-Evans rats, whereas short-term E2 lowered blood pressure and reduced renal damage. These findings highlight that the duration of hormone therapy may be an important factor for renal health in aging postmenopausal women.

GPER activation ameliorates aortic remodeling induced by salt-sensitive hypertension.

The mRen2 female rat is an estrogen- and salt-sensitive model of hypertension that reflects the higher pressure and salt sensitivity associated with menopause. We previously showed that the G protein-coupled estrogen receptor (GPER) mediates estrogenic effects in this model. The current study hypothesized that GPER protects against vascular injury during salt loading. Intact mRen2 female rats were fed a normal (NS; 0.5% Na(+)) or high-salt diet (HS; 4% Na(+)) for 10 wk, which significantly increased systolic blood pressure (149 ± 5 vs. 224 ± 8 mmHg;P< 0.001). Treatment with the selective GPER agonist G-1 for 2 wk did not alter salt-sensitive hypertension (216 ± 4 mmHg;P> 0.05) or ex vivo vascular responses to angiotensin II or phenylephrine (P> 0.05). However, G-1 significantly attenuated salt-induced aortic remodeling assessed by media-to-lumen ratio (NS: 0.43; HS+veh: 0.89; HS+G-1: 0.61;P< 0.05). Aortic thickening was not accompanied by changes in collagen, elastin, or medial proliferation. However, HS induced increases in medial layer glycosaminoglycans (0.07 vs. 0.42 mm(2);P< 0.001) and lipid peroxidation (0.11 vs. 0.51 mm(2);P< 0.01), both of which were reduced by G-1 (0.20 mm(2)and 0.23 mm(2); both P< 0.05). We conclude that GPER's beneficial actions in the aorta of salt-loaded mRen2 females occur independently of changes in blood pressure and vasoreactivity. GPER-induced attenuation of aortic remodeling was associated with a reduction in oxidative stress and decreased accumulation of glycosaminoglycans. Endogenous activation of GPER may protect females from salt- and pressure-induced vascular damage.