Overexpressing Transgenic Mice Research Articles

TXNIP regulates pulmonary inflammation induced by Asian sand dust

Asian sand dust (ASD), a seasonal dust storm originating from the deserts of China and Mongolia, affects Korea and Japan during the spring, carrying soil particles and a variety of biochemical components. Exposure to ASD has been associated with the onset and exacerbation of respiratory disorders, although the underlying mechanisms remain unclear. This study investigates ASD-induced pulmonary toxicity and its mechanistic pathways, focusing on the role of thioredoxin-interacting protein (TXNIP). Using TXNIP knock-out (KO) mice and adeno-associated virus (AAV)-mediated TXNIP overexpression transgenic mice, we explored how TXNIP modulates ASD-induced pulmonary inflammation. Mice were exposed to ASD via intranasal administration on days 1, 3, and 5 to induce inflammation. ASD exposure led to significant pulmonary inflammation, evidenced by increased inflammatory cell counts and elevated cytokine levels in bronchoalveolar lavage fluid, as well as heightened protein expression of the TXNIP/NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. TXNIP KO mice exhibited attenuated airway inflammation and downregulation of the NLRP3 inflammasome compared to wild-type controls, while AAV-mediated TXNIP overexpression mice showed exacerbated inflammatory responses, including elevated NLRP3 inflammasome expression, compared to AAV-GFP controls. These findings suggest that TXNIP is a key regulator of ASD-induced pulmonary inflammation.

Activation of Heme Metabolism Promotes Tissue Health After Intraarticular Injury or Surgical Exposure.

Posttraumatic osteoarthritis (PTOA) is a well-recognized public health burden without any disease modifying treatment. This occurs despite noted advances in surgical care in the past 50 years. Mitochondrial oxidative damage pathways initiate PTOA after severe injuries like intraarticular fracture that often require surgery and contribute to PTOA after less severe injuries that may or may not require surgery like meniscal injuries. When considering the mitochondrial and redox environment of the injured joint, we hypothesized that activation of heme metabolism, previously associated with healing in many settings, would cause prototypic mitochondrial reprogramming effects in cartilage ideally suited for use at the time of injury repair. Activation of heme metabolism can be accomplished through the gasotransmitter carbon monoxide (CO), which activates hemeoxygenase-1 (HO1) and subsequent heme metabolism. In this study, we employed unique carbon monoxide (CO)-containing foam (COF) to stimulate heme metabolism and restore chondrocyte oxygen metabolism in vitro and in vivo . Doxycycline-inducible, chondrocyte-specific HO1 overexpressing transgenic mice show similar mitochondrial reprogramming after induction compared to COF. CO is retained at least 24 h after COF injection into stifle joints and induces sustained increases in heme metabolism. Lastly, intraarticular injection of COF causes key redox outcomes without any adverse safety outcomes in rabbit stifle joints ex vivo and in vivo . We propose that activation of heme metabolism is an ideal adjuvant to trauma care that replenishes chondrocyte mitochondrial metabolism and restores redox homeostasis.

CSF1-dependent macrophage support matrisome and epithelial stress-induced keratin remodeling in Eosinophilic esophagitis

Atopic diseases such as Eosinophilic Esophagitis (EoE) often progress into fibrosis (FS-EoE), compromising organ function with limited targeted treatment options. Mechanistic understanding of FS-EoE progression is confounded by the lack of preclinical models and the heavy focus of research on eosinophils themselves. We found that macrophage accumulation precedes esophageal fibrosis in FS-EoE patients. We developed a FS-EoE model via chronic administration of oxazalone allergen, in a transgenic mouse over-expressing esophageal epithelial hIL-5 (L2-IL5OXA). These mice display striking histopathologic features congruent with that found in FS-EoE patients. Unbiased proteomic analysis, using a unique extracellular-matrix (ECM) focused technique, identified an inflammation-reactive provisional basal lamina membrane signature and this was validated in two independent EoE patient RNA-sequencing/proteomic cohorts, supporting model significance. A wound healing signature was also observed involving hemostasis-associated molecules previously unnoted in EoE. We further identified the ECM glycoprotein, Tenascin-C (TNC), and the stress-responsive keratin-16 (KRT16) as IL-4 and IL-13 responsive mediators, acting as biomarkers of FS-EoE. To mechanistically address how the immune infiltrate shapes FS-EoE progression, we phenotyped the major immune cell subsets that coalesce with fibrosis in both the L2-IL5OXA mice and in FS-EoE patients. We found that macrophage are required for matrisome and cytoskeletal remodeling. Importantly, we show that macrophage accumulation precedes esophageal fibrosis and provide a novel therapeutic target in FS-EoE as their depletion with anti-CSF1 attenuated reactive matrisome and cytoskeletal changes. Thus, macrophage-based treatments and the exploration of TNC and KRT16 as biomarkers may provide novel therapeutic options for patients with fibrostenosis.

Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8+ T cell recruitment

BackgroundLeukocyte immunoglobulin-like receptor B4 (LILRB4) plays a significant role in regulating immune responses. LILRB4 in microglia might influence the infiltration of peripheral T cells. However, whether and how LILRB4 expression aggravates brain damage after acute ischemic stroke remains unclear. This study investigates the role of LILRB4 in modulating the immune response and its potential protective effects against ischemic brain injury in mice.Methods and resultsMicroglia-specific LILRB4 conditional knockout (LILRB4-KO) and overexpression transgenic (LILRB4-TG) mice were constructed by a Cre-loxP system. Then, they were used to investigate the role of LILRB4 after ischemic stroke using a transient middle cerebral artery occlusion (tMCAO) mouse model. Spatial transcriptomics analysis revealed increased LILRB4 expression in the ischemic hemisphere. Single-cell RNA sequencing (scRNA-seq) identified microglia-cluster3, an ischemia-associated microglia subcluster with elevated LILRB4 expression in the ischemic brain. Flow cytometry and immunofluorescence staining showed increased CD8+ T cell infiltration into the brain in LILRB4-KO-tMCAO mice. Behavioral tests, cortical perfusion maps, and infarct size measurements indicated that LILRB4-KO-tMCAO mice had more severe functional deficits and larger infarct sizes compared to Control-tMCAO and LILRB4-TG-tMCAO mice. T cell migration assays demonstrated that LILRB4-KD microglia promoted CD8+ T cell recruitment and activation in vitro, which was mitigated by CCL2 inhibition and recombinant arginase-1 addition. The scRNA-seq and spatial transcriptomics identified CCL2 was predominantly secreted from activated microglia/macrophage and increased CCL2 expression in LILRB4-KD microglia, suggesting a chemokine-mediated mechanism of LILRB4.ConclusionLILRB4 in microglia plays a crucial role in modulating the post-stroke immune response by regulating CD8+ T cell infiltration and activation. Knockout of LILRB4 exacerbates ischemic brain injury by promoting CD8+ T cell recruitment. Overexpression of LILRB4, conversely, offers neuroprotection. These findings highlight the therapeutic potential of targeting LILRB4 and its downstream pathways to mitigate immune-mediated damage in ischemic stroke.

IL-10 Overexpression Reduces the Protective Response of an Experimental Chlamydia abortus Vaccine in a Murine Model.

In ovine populations, the enzootic nature of Chlamydia abortus (C. abortus) is attributed to its capacity to establish persistent intracellular infections, which necessitate a cellular immune response mediated by interferon-gamma (IFN-γ) for effective resolution. In both natural hosts and murine models, interleukin-10 (IL-10) has been demonstrated to modulate the cellular immune response crucial for the eradication of C. abortus. During gestation, it has also been shown to play a role in preventing inflammatory damage to gestational tissues and foetal loss through the downregulation of pro-inflammatory cytokines. This paradigm can be key for events leading to a protective response towards an infectious abortion. Previous research successfully established a mouse model of chronic C. abortus infection using transgenic mice overexpressing IL-10 (IL-10tg), simulating the dynamics of chronic infection observed in non-pregnant natural host. This study aims to evaluate the efficacy of an experimental inactivated vaccine against C. abortus and to elucidate the immune mechanisms involved in protection during chronic infection using this model. Transgenic and wild-type (WT) control mice were immunized and subsequently challenged with C. abortus. Vaccine effectiveness and immune response were assessed via immunohistochemistry and cytokine serum levels over a 28-day period. Morbidity, measured by daily weight loss, was more pronounced in non-vaccinated transgenic IL-10 mice, though no mortality was observed in any group. Vaccinated control mice eliminated the bacterial infection by day 9 post-infection (p.i.), whereas presence of bacteria was noted in vaccinated transgenic IL-10 mice until day 28 p.i. Vaccination induced an early post-infection increase in IFN-γ production, but did not alter IL-10 production in transgenic mice. Histological analysis indicated suboptimal recruitment of inflammatory cells in vaccinated transgenic IL-10 mice compared to WT controls. In summary, the findings suggest that IL-10 overexpression in transgenic mice diminishes the protective efficacy of vaccination, confirming that this model can be useful for validating the efficacy of vaccines against intracellular pathogens such as C. abortus that require robust cell-mediated immunity.

Abstract We106: Long Non-Coding RNA LIPTER Regulates Lipid Metabolism of Human Cardiomyocytes and Preserves Cardiac Function

Background: Recently, long non-coding RNAs (lncRNAs) have emerged as novel human-specific (i.e. non-conserved) regulators of cardiovascular development and disease, which possess diverse functions through interacting with other molecules, including DNA, RNA, protein, and lipid. We identified a human lncRNA, LIPTER, which mediates lipid droplet (LD) transport to regulate lipid metabilism of human cardiomyocytes (CMs). Mechanistically, LIPTER binds phospholipids PA and PI4P on the LD membranes and the MYH10 protein, connecting LDs to the cytoskeleton and facilitating LD transport to mitochondria. LIPTER deficiencies led to prominent LD accumulation, mitochondrial dysfunction, and death of human iPS cell-derived CMs. Since downregulation of LIPTER and enahnced LD accumulation are associated with cardiac dysfunction and heart failure in patients with metabolic disorders, such as obesity and diabetes mellitus, we thereby seek to explore the preclinical potential of LIPTER for the therapy of human metabolic disorder associated cardiac dysfunction and heart failure. Hypothesis: We hypothesize that transgenic overexpression of the human lncRNA LIPTER in mouse heart could attenuate metabolic syndrome-associated myocardium lipid accumulation and cardiac dysfunction, as well as prevent isoproterenol-induced heart failure through increasing the fatty acid oxidation (FAO) of mouse heart. Goals: To evaluate the preclinical potential of human lncRNA LIPTER for the therapy of cardiac dysfunction and heart failure by using mouse cardiac dysfunction and heart failure models. Methods: We established a LIPTER transgenic overexpression mouse line (LIPTER Tg ). Both WT and LIPTER Tg mice were fed with high fat diet for 10 months to induce cardiac lipid accumulation and dysfunction. Additionally, heart failure was induced by the chronic isoproterenol administration. After treatment, mice were subjected to histological and molecular analyses. Results: We found LIPTER Tg significantly enhanced FAO of mouse heart, reduced cardiac lipid accumulation and fibrosis, and alleviated cardiac dysfunction of high-fat-diet fed mice, as well as preserved cardiac function of mice post isoproterenol administration. Conclusions: We unveil a crucial role of human lncRNA LIPTER in regulating lipid metabolism of human CMs and testify its clinical potential for treating cardiac dysfunction and heart failure.

Selenoprotein W modulates tau homeostasis in an Alzheimer’s disease mouse model

Lower selenium levels are observed in Alzheimer’s disease (AD) brains, while supplementation shows multiple benefits. Selenoprotein W (SELENOW) is sensitive to selenium changes and binds to tau, reducing tau accumulation. However, whether restoration of SELENOW has any protective effect in AD models and its underlying mechanism remain unknown. Here, we confirm the association between SELENOW downregulation and tau pathology, revealing SELENOW’s role in promoting tau degradation through the ubiquitin‒proteasome system. SELENOW competes with Hsp70 to interact with tau, promoting its ubiquitination and inhibiting tau acetylation at K281. SELENOW deficiency leads to synaptic defects, tau dysregulation and impaired long-term potentiation, resulting in memory deficits in mice. Conversely, SELENOW overexpression in the triple transgenic AD mice ameliorates memory impairment and tau-related pathologies, featuring decreased 4-repeat tau isoform, phosphorylation at Ser396 and Ser404, neurofibrillary tangles and neuroinflammation. Thus, SELENOW contributes to the regulation of tau homeostasis and synaptic maintenance, implicating its potential role in AD.

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

LncRNA CCRR maintains Ca2+ homeostasis against myocardial infarction through the FTO-SERCA2a pathway.

Cardiac conduction regulatory RNA (CCRR) has been documented as an antiarrhythmic lncRNA in our earlier investigation. This study aimed to evaluate the effects of CCRR on SERCA2a and the associated Ca2+ homeostasis in myocardial infarction (MI). Overexpression of CCRR via AAV9-mediated delivery not only partially reversed ischemia-induced contractile dysfunction but also alleviated abnormal Ca2+ homeostasis and reduced the heightened methylation level of SERCA2a following MI. These effects were also observed in CCRR over-expressing transgenic mice. A conserved sequence domain of CCRR mimicked the protective function observed with the full length. Furthermore, silencing CCRR in healthy mice led to intracellular Ca2+ overloading of cardiomyocytes. CCRR increased SERCA2a protein stability by upregulating FTO expression. The direct interaction between CCRR and FTO protein was characterized by RNA-binding protein immunoprecipitation (RIP) analysis and RNA pulldown experiments. Activation of NFATc3 was identified as an upstream mechanism responsible for CCRR downregulation in MI. This study demonstrates that CCRR is a protective lncRNA that acts by maintaining the function of FTO, thereby reducing the m6A RNA methylation level of SERCA2a, ultimately preserving calcium homeostasis for myocardial contractile function in MI. Therefore, CCRR may be considered a promising therapeutic strategy with a beneficial role in cardiac pathology.

Angiotensin II-induced cardiac dysfunction and fibrosis is prevented by cardiomyocyte-specific overexpression of Larp6

Introduction: La ribonucleoprotein 6, Translational Regulator (Larp6) is a multifunctional mRNA binding protein most well described for its pro-fibrotic action to increase type I collagen mRNA half-life, translation, and deposition. In the heart, the actions of Larp6 are not described. Interestingly, Larp6 is expressed in cardiomyocytes which are not primarily involved in cardiac fibrosis. To investigate the role of cardiomyocyte Larp6 in cardiac function and remodeling, we generated a cardiomyocyte-specific Larp6 overexpressing (OE) transgenic mouse model and determined its role at (i) baseline and (ii) following hypertensive challenge with angiotensin II (Ang II). We hypothesized that cardiomyocyte-specific Larp6 OE exacerbates Ang II-induced cardiac remodeling and dysfunction. Methods: Cross-sectional studies were performed from 2-8 months of age in male and female Larp6 OE and wildtype (WT) littermates. Baseline cardiac function was assessed by 7-T MRI and echocardiography. In a separate set of mice (10 mo of age), hypertension was induced via Ang II infusion by osmotic minipump (1000 ng/kg/min) or vehicle. Blood pressure (BP) was assessed 14-17 days post-infusion via tail-cuff plethysmography, cardiac function by echocardiography after 21 days, and the heart prepared for histochemistry. Differences in endpoints were determined by two-way ANOVA with Tukey post hoc and a significant p-value cutoff of 0.05. Results: Baseline studies revealed Larp6 OE had no significant effect on cardiac function (7-T MRI), BP, or gross morphology in either sex across timepoints. At 10 months of age, histochemistry revealed increased cardiomyocyte Larp6 protein expression and mild, but significant, interstitial fibrosis in unstressed Larp6 OE mice. In 10-month-old mice, Ang II infusion similarly increased BP and induced cardiac hypertrophy in both male and female Larp6 OE and WT mice. Further, Ang II induced systolic dysfunction and cardiac fibrosis in WT mice. Unexpectedly, Larp6 OE prevented Ang II-mediated interstitial fibrotic remodeling and decreases in ejection fraction, fractional shortening, and cardiac output independent of sex. Of note, Larp6 OE did not protect against Ang II-induced perivascular fibrosis, suggesting the anti-fibrotic effect was localized around the cardiomyocytes. Conclusion: Taken together, these data support a pro-fibrotic action of cardiomyocyte Larp6 in unstressed mice. Despite this impact at baseline, cardiomyocyte-specific Larp6 OE is suffcient to prevent Ang II-induced cardiac fibrosis and dysfunction, independent of BP and sex. This unexpected result, contrary to our hypothesis, suggests that targeting cardiomyocyte Larp6 signaling may represent a new therapeutic avenue to treat hypertensive heart disease. R01 HL136386, NIDDK DK130243, SRCS (IK6BX004016), BX005845, and R21 AA029747. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Netrin-1 increases epithelial cell proliferation and promotes renal cyst formation by activating Rac2 in collecting duct cells

Despite recent advances in our understanding of the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. Common features of PKD are excessive epithelial cell proliferation and fluid-filled cyst formation in the kidney, leading to loss of kidney function. We recently demonstrated that netrin-1 is upregulated in primary human PKD cystic cells and in the kidneys of rodent models of PKD. Moreover, overexpression of netrin-1 in tubular epithelium increases cell proliferation (p<0.01) and induces renal cyst development (p<0.0001), while decreasing netrin-1 reduces cyst growth (p<0.01) and improves kidney function (p<0.05). However, the underlying mechanism by which netrin-1 induces cystic cell proliferation is unknown. We hypothesize that netrin-1 increases cystic epithelial cell proliferation by activating Ras-Related C3 Botulinum Toxin Substrate 2 (Rac2) in collecting duct (CD). 4-week-old male tubular overexpressed netrin-1 transgenic mice (NTN-1 Tg6) and 8-week old Pkd1RC/RC mice were randomized to receive vehicle or the specific Rac2 inhibitor EHT 1864 (20 mg/kg/48hr i. p; n=4-5/group) for 4 weeks. Blood and kidneys were collected for biochemical, histological, and Western blot analysis. Renal co-localization of Rac2 with CD specific marker Auaphorin-2 in kidney of human and mouse PKD was assessed by immunofluorescence staining and protein expression was measured by Western blot analysis. Renal Rac2 activity was measured using Rac2 activation kit. Cystic index were measured using H&E staining. Cell proliferation was measured using MTT assay and renal function was measured using BUN assay kit. Rac2 is upregulated in the CD of both human PKD and Pkd1RC/RC mice (p<0.01) compared to controls. Moreover, overexpression of netrin-1 activates Rac2 in the cystic kidney (p<0.001) and in human cystic cells (p<0.05). Overexpression of Rac2 increases cell proliferation and phosphorylation of ERK in CD cells (p<0.01). Inhibition of Rac2 reduces netrin-1-induced CD cell proliferation (p<0.01) and cyst growth (p<0.01) in both netrin-1 transgenic (NTN-1 Tg6) and Pkd1RC/RC mice. Our results demonstrate that overexpression of netrin-1 increases cystic epithelial cell proliferation via activating Rac2 in CD cells and contributes to cyst growth. Future studies will be carried out to dissect the downstream signaling mechanism of netrin-1/Rac2 mediated cell proliferation cyst formation in PKD. This work is supported by Intramural grant award (IGPP00043) from Augusta University to R. Mohamed; National Institute of Health (1P01HL134604-01) to J.C. Sullivan. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

ACE2 Rescues Sepsis-Associated Encephalopathy by Reducing Inflammation, Oxidative Stress, and Neuronal Apoptosis via the Nrf2/Sestrin2 Signaling Pathway.

Neuroinflammation and oxidative stress contribute to the progression of sepsis-associated encephalopathy (SAE). Angiotensin-converting enzyme 2 (ACE2) is considered to be a neuroprotective factor due to its anti-inflammatory and antioxidant properties. However, the role of ACE2 on myeloid cells in regulating SAE and the underlying mechanism warrants further exploration. SAE was induced in ACE2 transgenic (TG), knockout (KO), and bone marrow (BM) chimeric mice by cecal ligation and puncture (CLP). The expression levels of apoptosis-, oxidation- and neuroinflammation-associated mediators and morphological changes were monitored by quantitative real-time PCR analyses and histological examinations in the cortex of septic mice. The contents of angiotensin (Ang) II and Ang-(1-7) along with the activity of ACE2 were examined with commercial kits. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Sestrin2 was detected by immunoblotting analysis. Our results indicated that the expression of cortical ACE2 was significantly reduced in the early phase of CLP-induced sepsis. Moreover, ACE2 overexpression in TG mice conferred neuroprotection against sepsis, as evidenced by alleviated neuronal apoptosis, oxidative stress, and proinflammatory M1-like microglial polarization, accompanied by upregulation of the Ang-(1-7), Nrf2, and Sestrin2 protein levels. Conversely, ACE2 deficiency in KO mice exacerbated SAE. The neuroprotective effects of ACE2 were further confirmed in wild-type mice transplanted with ACE2-TG and KO BM cells. Therefore, our data suggest that myeloid ACE2 exerts a protective role in the pathogenesis of SAE, potentially by activating Ang-(1-7)-Nrf2/sestrin2 signaling pathway, and highlight that upregulating ACE2 expression and activity may represent a promising approach for the treatment of SAE in patients with sepsis.

Effect of prothymosin α on neuroplasticity following cerebral ischemia‑reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti‑necrotic and anti‑apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia‑reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen‑glucose deprivation for 2h invitro. Immunofluorescence staining for ProT and MAP‑2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50min to evaluate the effect of ProT on neuroplasticity‑associated protein following ischemia‑reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain‑derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal‑associated protein, 25kDa and postsynaptic density protein 95 after ischemic‑reperfusion injury invivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia‑reperfusion injury.

Abstract A084: DEK overexpression in the murine mammary gland promotes epithelial hyperplasia

Abstract Introduction: Breast cancer (BC) remains a leading cause of cancer-associated mortality for women in the USA, which makes it critically important to identify and study molecular drivers of pathogenesis. We have previously identified the chromatin modifying DEK protein to have oncogenic properties. DEK is a histone H3 chaperone that is over-expressed in over 60% of breast cancers, particularly triple negative breast cancers, and is associated with poor survival. To date, all studies regarding the role of DEK in BC have manipulated DEK expression in cultured immortalized MCF10A human mammary epithelial cells and BC cell lines. However, the oncogenic functions of DEK in vivo and in normal mammary epithelium have remained unexplored. Methods: We utilized a tetracycline-off regulated, DEK overexpressing (DEK-OE) transgenic mouse model that targets mammary epithelium through the mouse mammary tumor virus (MMTV) promoter. We subsequently performed bulk RNA-Seq transcriptomic analyses, western blotting, and both histological and morphological assessments of control vs. DEK-OE mammary gland tissue to identify the oncogenic effects of DEK over-expression in vivo. Results: Bulk RNA sequencing and gene ontology analyses identified cell cycle regulation as a significantly upregulated biological process in DEK-OE versus control glands. Subsequent studies confirmed upregulation of proteins that promote the G1-S cell cycle transition including cyclins A and D and CDKs 2, 4, and 6 as well as increased numbers of Ki67+ cells. We also observed down-regulation of genes that inhibit entry the G1-S transition, such as p21 and p27. This pro-proliferative molecular status correlated with the development of mammary gland hyperplasia. Importantly, in silico analyses demonstrated gene correlations between DEK and cell cycle regulators that were also evident in human breast cancers. One potential mechanism for this transcriptional deregulation is that we identified increased histone H3K27me3 epigenetic modifications in DEK-OE cells, in both mouse and human models, which may occur through EZH2 activity. Conclusions: Combined, our work indicates that DEK is not a tumor initiator, but its oncogenic activities are likely due to its ability to promote proliferation of mammary epithelial cells. Furthermore, we report a novel function for DEK in the epigenetic regulation of histone H3 K27 trimethylation, which is mediated by the well-known EZH2 oncogene in triple negative breast cancers. Citation Format: Megan Johnstone, Ashley Leck, Taylor Lange, Lisa Privette Vinnedge. DEK overexpression in the murine mammary gland promotes epithelial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A084.

Chronic stress dysregulates the Hippo/YAP/14-3-3η pathway and induces mitochondrial damage in basolateral amygdala in a mouse model of depression.

Rationale: Recent evidence highlights the pivotal role of mitochondrial dysfunction in mood disorders, but the mechanism involved remains unclear. We studied whether the Hippo/YAP/14-3-3η signaling pathway mediates mitochondrial abnormalities that result in the onset of major depressive disorder (MDD) in a mouse model. Methods: The ROC algorithm was used to identify a subpopulation of mice that were exposed to chronic unpredictable mild stress (CUMS) and exhibited the most prominent depressive phenotype (Dep). Electron microscopy, biochemical assays, quantitative PCR, and immunoblotting were used to evaluate synaptic and mitochondrial changes in the basolateral amygdala (BLA). RNA sequencing was used to explore changes in the Hippo pathway and downstream target genes. In vitro pharmacological inhibition and immunoprecipitation was used to confirm YAP/14-3-3η interaction and its role in neuronal mitochondrial dysfunction. We used virus-mediated gene overexpression and knockout in YAP transgenic mice to verify the regulatory effect of the Hippo/YAP/14-3-3η pathway on depressive-like behavior. Results: Transcriptomic data identified a large number of genes and signaling pathways that were specifically altered from the BLA of Dep mice. Dep mice showed notable synaptic impairment in BLA neurons, as well as mitochondrial damage characterized by abnormal mitochondrial morphology, compromised function, impaired biogenesis, and alterations in mitochondrial marker proteins. The Hippo signaling pathway was activated in Dep mice during CUMS, and the transcriptional regulatory activity of YAP was suppressed by phosphorylation of its Ser127 site. 14-3-3η was identified as an important co-regulatory factor of the Hippo/YAP pathway, as it can respond to chronic stress and regulate cytoplasmic retention of YAP. Importantly, the integrated Hippo/YAP/14-3-3η pathway mediated neuronal mitochondrial dysfunction and depressive behavior in Dep mice. Conclusion: The integrated Hippo/YAP/14-3-3η pathway in the BLA neuron is critical in mediating depressive-like behaviors in mice, suggesting a causal role for this pathway in susceptibility to chronic stress-induced depression. This pathway therefore may present a therapeutic target against mitochondrial dysfunction and synaptic impairment in MDD.

Elevation of CISD2 attenuates atrial aging

Abstract Background Age-related atrial cardiomyopathy is characterized by conduction disturbance, structural fibrosis as well as cardiac remodeling. Atrial cardiomyopathy promotes the development of atrial fibrillation, which is associated with a 5-fold increase in the risk of stroke. However, the molecular mechanisms underlying age-associated deterioration of atrial function remain unclear. Cisd2 is a mitochondrial outer membrane protein that regulates intracellular calcium homeostasis and mitochondrial integrity. Importantly, Cisd2 is a pro-longevity gene in mammals. Purpose In this study, we investigate the role of Cisd2 in atrial cardiomyopathy during aging. Methods CISD2 expression and atrial conductance were examined in different age human surgical samples. Atrial conductance was assessed and validated by immunofluorescence and Transmission electron microscope in Cisd2 knockout and overexpressing transgenic mice at young and old age. The mitochondrial functions and calcium homeostasis were investigated in the atrial HL-1 cell lines with Cisd2KO or re-expression. Transcriptomic analysis of mice atrial tissues was performed. Results The age-associated decrease of atrial Cisd2 protein, in human and mice, were associated with atrial conductance disturbance and intercalated disc disorganization. Furthermore, Cisd2 deficiency disrupts calcium homeostasis via dysregulation of Serca2a and Stim1 resulting in an increased level of basal cytosolic Ca2+ and mitochondrial Ca2+ overload in HL-1 atrial cardiomyocytes. Most strikingly, in Cisd2 transgenic mice, a persistently high level of Cisd2 is sufficient to delay age-associated atrial myopathy and attenuate age-related atrial structural defects and functional decline. In addition, it results in a younger atrial transcriptome pattern during old age. Conclusions The mitochondrial-associated membrane-bounded CDGSH iron-sulfur domain-containing protein 2 (CISD2) protein regulates cytosolic calcium homeostasis and mitochondrial integrity. Interestingly, an age-dependent decrease of atrial Cisd2 protein has been detected during aging in both humans and mice. Our findings indicate that decreased atrial Cisd2 is one of the causes of atrial myopathy during cardiac aging. A persistently high level of atrial Cisd2 delays atrial myopathy during aging and ameliorates age-related atrial dysfunction.

Molecular Features and Stages of Pulmonary Fibrosis Driven by Type 2 Inflammation.

Systemic sclerosis (SSc) is a progressive, multiorgan disease with limited treatment options. Although a recent proof-of-concept study using romilkimab or SAR156597, a bispecific IL-4/IL-13 antibody, suggests a direct role of these cytokines in the pathophysiology of SSc, their contributions to the balance between inflammation and fibrosis are unclear. Here, we determine the roles of type 2 inflammation in fibrogenesis using FRA2-Tg (Fos-related antigen 2-overexpressing transgenic) mice, which develop spontaneous, age-dependent progressive lung fibrosis. We defined the molecular signatures of inflammation and fibrosis at three key stages in disease progression, corresponding to preonset, inflammatory dominant, and fibrosis dominant biology, and revealed an early increase in cytokine-cytokine receptor interactions and antigen-processing and presentation pathways followed by enhanced Th2- and M2 macrophage-driven type 2 responses. This type 2 inflammation progressed to extensive fibrotic pathology by 14-18 weeks of age, with these gene signatures overlapping significantly with those seen in the lungs of patients with SSc with interstitial lung disease (ILD). These changes were also evident in the histopathology, which showed perivascular and peribronchiolar inflammation with prominent eosinophilia and accumulation of profibrotic M2-like macrophages followed by rapid progression to fibrosis with thickened alveolar walls with multifocal fibrotic bands and signs of interstitial pneumonia. Critically, treatment with a bispecific antibody targeting IL-4 and IL-13 during the inflammatory phase abrogated the Th2 and M2 responses and led to near-complete abrogation of lung fibrosis. These data recapitulate important features of fibrotic progression in the lungs of patients with SSc-ILD and enhance our understanding of the progressive pathobiology of SSc. This study also further establishes FRA2-Tg mice as a valuable tool for testing future therapeutic agents in SSc-ILD.

Sustained Increases in Cardiomyocyte Protein O-Linked β-N-Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment.

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked β-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.

Impact of common ALDH2 inactivating mutation and alcohol consumption on Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is an enzyme found in the mitochondrial matrix that plays a central role in alcohol and aldehyde metabolism. A common ALDH2 polymorphism in East Asians descent (called ALDH2*2 or E504K missense variant, SNP ID: rs671), present in approximately 8% of the world's population, has been associated with a variety of diseases. Recent meta-analyses support the relationship between this ALDH2 polymorphism and Alzheimer's disease (AD). And AD-like pathology observed in ALDH2-/- null mice and ALDH2*2 overexpressing transgenic mice indicate that ALDH2 deficiency plays an important role in the pathogenesis of AD. Recently, the worldwide increase in alcohol consumption has drawn attention to the relationship between heavy alcohol consumption and AD. Of potential clinical significance, chronic administration of alcohol in ALDH2*2/*2 knock-in mice exacerbates the pathogenesis of AD-like symptoms. Therefore, ALDH2 polymorphism and alcohol consumption likely play an important role in the onset and progression of AD. Here, we review the data on the relationship between ALDH2 polymorphism, alcohol, and AD, and summarize what is currently known about the role of the common ALDH2 inactivating mutation, ALDH2*2, and alcohol in the onset and progression of AD.