Biology subjects

Machado, R. F.

Publications and source records attributed to Machado, R. F..

7 recordsLinked to original sources

bioRxiv2026

Hemin Triggers Cytoprotective Lipid Droplet Formation in Pulmonary Microvascular Endothelial Cells

Sickle cell disease (SCD) is characterized by chronic and episodic hemolysis, inducing endothelial cell dysfunction and death. Lipid droplets (LDs) accumulate intracellularly in response to stress but the role of LDs in hemin-stressed pulmonary microvascular cells remain unexplored. We hypothesized that LD formation is an adaptive response that improves endothelial cell survival during hemin-induced stress. We utilized cultures of human lung microvascular endothelial cells (hLMVECs) to test this hypothesis. Hemin alone was able to induce LD biogenesis in culture. In response to hemin treatment, we found an increase in cellular death by lactate dehydrogenase (LDH) assay when LD biogenesis was inhibited with a small molecule inhibitor of diacylglycerol acyltransferase 1 (DGAT1), A922500. Induction of LDs by treatment with oleic acid (OA) coupled with hemin resulted in improved endothelial cell survival. Hemin treatment was able to activate p-JNK and p-P38 signaling, while treatment with OA alone could not induce either signaling pathway. Hemin treatment combined with DGAT1 inhibition resulted in an increase in endothelial cell secreted IL-6 and IL-8 levels as measured by enzyme linked immunosorbent assay (ELISA), demonstrating an increase in endothelial cell dysfunction and stress. Induction of LD formation with oleic acid (OA) treatment resulted a reduction in secreted IL-6 and IL-8. Mitochondrial reactive oxygen species (ROS) production was decreased by OA treatment, as evidenced by MitoSOXRed assay. These data suggest that hLMVECs are protected from hemin-induced inflammation and death by the presence of LDs. Further studies are needed to explore the Hemin-LD-Cell Death axis for translational therapeutic use.

physiology↗

bioRxiv2026

In silico structural analysis of EthA substitutions for ranking priority mutations leading to ethionamide resistance in Mycobacterium tuberculosis

BackgroundTuberculosis (TB) is the second-leading cause of deaths from infectious agents and remains a global health threat. Ethionamide (ETH) is a prodrug used in regimens for multidrug-resistant TB, and, partly due to side effects that can lead to low treatment adhesion, resistance arises. Changes in EthA, the monooxygenase that activates ETH, are the main mechanism of resistance. Yet, of hundreds of EthA substitutions found in resistant isolates, only a handful have been annotated as resistance determinants. ResultsAn in silico analysis was carried out on a previously described panel of Mycobacterium tuberculosis clinical isolates for which genomes and ETH susceptibility testing results were available. EthA substitutions were mapped, revealing the existence of hotspots in its sequence. Visualization of the hotspots in the EthA structural model shows that they cluster in three regions, including ligand binding pockets. Models were built of twenty-three variants found in resistant isolates and changes in local configuration was mapped to identify investigate impact on ETH activation. Information from these models contributed to establishing five criteria for scoring whether substitutions are most likely to lead to resistance. Using these criteria, EthA D58G was selected and its expression is shown to increase growth in high ETH concentrations. ConclusionFunctionally relevant regions of EthA are revealed and point out priority substitutions for functional studies, enhancing identification and detection of substitutions not been previously associated with resistance.

molecular biology↗

bioRxiv2026

Nicotinamide phosphoribosyltransferase activates the mitochondrial unfolded protein response to promote pulmonary arterial endothelial cell proliferation

Vascular remodeling leading to occlusion of the pulmonary artery and increased pulmonary vascular resistance is the central feature of Pulmonary Arterial Hypertension (PAH) which leads to death due to right heart failure. The expression of Nicotinamide phosphoribosyltransferase (Nampt) is increased in the lungs and isolated PAECs of PAH patients. Inhibition of NAMPT is protective in preclinical models of pulmonary hypertension. NAMPT regulates multiple mitochondrial processes that control cell proliferation and survival. Using rodent models of PH, we demonstrated that the mitochondrial unfolded protein response (UPRmt) promotes vascular remodeling. Hence we, hypothesized that NAMPT activates the UPRmt to promote abnormal pulmonary arterial endothelial cell proliferation. Analysis of PAECs isolated from PAH patients show increased expression of the UPRmt pathway mediators, ATF-5, mtHSP70 and ClpP. Human PAEC cell lines were exposed to recombinant Nampt or transduced with lentiviral-Nampt. We observed increased phosphorylation and activation of eIF2 which permits preferential translation of the ATF-5 transcription factor. ATF-5 expression and nuclear localization was increased. Further, we observed increased expression of ATF-5 target genes, mtHSP70, HSP60, ClpP and LonP1 as well as increased PAEC proliferation. Blocking mtHSP70 function reversed the Nampt induced increase in proliferation. Neither UPRmt activation nor proliferation was increased in the presence of enzymatically inactive-Nampt. Our observations demonstrate that Nampt can promote PAEC proliferation by activating the UPRmt and uncovers novel potential targets to address vascular remodeling in PAH.

cell biology↗

bioRxiv2025

Pulmonary Hypertension Promotes Neuroinflammation and Neurodegeneration

IntroductionPulmonary arterial hypertension (PAH) is associated with neurocognitive deficits and abnormal brain MRI. Little is known about the mechanisms underlying these clinical observations. TDP-43 is a proteinopathy associated with frontotemporal lobar degeneration (FTLD), Alzheimers Disease, and Amyotrophic Lateral Sclerosis (ALS). In this study, we hypothesize PAH will result in gliosis, reduced neuronal density, and increased TDP-43 mislocalization. MethodsSprague Dawley rats were randomly assigned to receive Vehicle (DMSO) Monocrotaline, or Sugen/Hypoxia to induce PH. Right heart catheterization was used to confirm PAH. Brain tissue was fixed and probed for microglia (Iba1), astrocytes (GFAP), neurons (NeuN), and TDP-43. Human PH vs control brain tissue was also probed for NeuN and TDP-43. Results and ConclusionsWe identify an increase in microglia and astrocyte density in the frontal cortex along with reduced neuronal density and neuronal TDP-43 mislocalization in rat models of PH. In addition, human PH frontal cortex demonstrated neuronal TDP-43 mislocalization. This is the first evidence of TDP-43 proteinopathy in PH.

physiology↗

bioRxiv2025

The mitochondrial unfolded protein response and metabolic reprogramming promote PASMC proliferation in response to sphingosine kinase-1/sphingosine-1-phosphate signaling

Proliferation and vasoconstriction of the intimal smooth muscle layer of the pulmonary artery are pathogenic characteristics of pulmonary arterial hypertension (PAH). Altered mitochondrial function, i.e. glycolysis, ROS generation and fission, are known potentiators of vascular remodeling. However, most current therapeutic interventions fail to effectively address the proliferation of the pulmonary artery smooth muscle cells (PASMCSs) lining the pulmonary vasculature and highlight the importance of identifying novel pathways to target for intervention. The Sphk1/S1P/S1P2 signaling axis is upregulated in PAH patients and is known to induce PASMC proliferation during hypoxia-mediated pulmonary hypertension (HPH). Interestingly, Sphk1 modulates mitochondrial function in that it regulates dynamics, cell growth and survival and in C. elegans, it induces activation of the unfolded protein response (UPRmt). We aimed to investigate if the Sphk1/S1P/S1P2 signaling axis promotes vascular remodeling in PAH via activation of the UPRmt. PASMCs isolated from IPAH patients were subjected to RNAseq analysis. The effect of Sphk1 or S1P was interrogated in hPASMC cell lines and the HPH model was used to assess the effect of the UPRmt on PAH pathogenesis. RNAseq analysis revealed that pathways involved in mitochondrial respiration were among the top 20 most significantly regulated pathways. Furthermore, ATF-5, the transcription factor that mediates the UPRmt was significantly upregulated. In hPASMCs, Sphk1/S1P lead to decreased respiration, increased glycolysis, fission, ROS and UPRmt activation. Pharmacological inhibition of the UPRmt mediator, mtHSP70, mitigated the Sphk1 induced increase in hPASMC proliferation. Furthermore, mtHSP70 inhibition was protective in hypoxia-mediated pulmonary hypertension (HPH) as we observed a decrease in right ventricular systolic pressure, right ventricular hypertrophy and vascular remodeling. These data suggest that the UPRmt promotes vascular remodeling in PAH and may present a novel pathway to target for pharmaceutical intervention.

cell biology↗

bioRxiv2023

Endothelial Knockdown of the Tumor Suppressor, WWOX, Increases Inflammation in Ventilator-Induced Lung Injury

BackgroundChronic cigarette smoke exposure downregulates lung expression of WWOX, an ARDS relevant tumor suppressor. Prior work has revealed a barrier protective function of WWOX during infectious models of ARDS. Proteomic analysis of WWOX-silenced lung endothelial cells suggest involvement of WWOX in protection against mechanical stretch-induced inflammation. MethodsProtein lysates from WWOX-silenced endothelial cells (ECs) were analyzed using tandem mass tag mass spectrometry (TMT-MS) to determine the differential expression status of the proteome compared to wild type ECs. WWOX-silenced ECs as well as those isolated from endothelial Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 hours). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. Dual silencing of WWOX and zyxin was achieved to determine the role of zyxin upregulation in IL-8 production following mechanical stretch and during WWOX knockdown. Control and EC Wwox KO mice were subjected to high tidal volume ventilation (VILI, 40ml/kg, 65 breath/min, 4hours). Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histologic assays. ResultsTMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1{beta}, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 MAPK phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion. ConclusionLoss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears to be dependent on upregulation of zyxin.

molecular biology↗

bioRxiv2020

Cigarette Smoke and Nicotine-Containing E-cigarette Vapor Downregulate Lung WWOX Expression Which is Associated with Increased Severity of Murine ARDS

RATIONALEA history of chronic cigarette smoking is known to increase risk for ARDS, but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures, and thus an interesting candidate gene for the study of exposure-related lung disease. METHODS AND RESULTSLungs harvested from current versus former/never smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle treated controls. In separate studies, endothelial (EC)-specific WWOX KO versus wild type mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+CD45- cells. These were grown in culture, confirmed to be WWOX-deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing (ECIS) as well as a FITC dextran transwell assay for their barrier properties during MRSA or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to wild type cells during either MRSA or LPS treatment as measured by both ECIS and the transwell assay. CONCLUSIONThe increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.

molecular biology↗