NIH Grant Research Articles

Mildronate ameliorates kidney injury and reduces inflammation in Sickle Cell Disease mice

INTRODUCTION: Sickle cell disease (SCD) is a hereditary hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and chronic inflammation that affects multiple organs including kidney, liver, brain, spleen, and heart. Renal disease, the most common complication in SCD, is a progressive disease that begins in childhood as glomerular hyperfiltration, then advances to albuminuria, following by a fast decline in glomerular filtration. It may further progress to a renal failure in adults. More than 50% of SCD patients develop chronic kidney disease (CKD), and 14-18% progress to the end-stage kidney disease. Renal injury in SCD results from a cascade of events starting with chronic red blood cells sickling and hemolysis leading to increased blood viscosity, microvascular obstruction and dilation, hypoxia in the medulla, oxidative stress, and inflammation. Mildronate (meldonium) is an anti-ischemic and anti-inflammatory medication approved in Eastern Europe. HYPOTHESIS: We hypothesized that mildronate administration reduces renal medullary hypoxia and decreases oxidative stress and inflammation ameliorating renal injury in SCD mice. METHODS: The study was approved by the Howard University IACUC. Townes SCD mice, expressing human HbS, and control mice, expressing human HbA, were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood was collected and used for hematological parameters analysis. Cytokines were measured by Bio-Plex suspension array in plasma. Hypoxyprobe Biotin kit (Hypoxyprobe Inc) was used for visualization of organ hypoxia. The effect of Mildronate on systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). Urinary KIM-1 was measured by TIM-1/KIM-1 ELISA kit (R&D Systems). RESULTS: Mildornate injections did not affect hematological parameters of SCD mice including hematocrit, MCV, MCH, CMCH, erythrocytes and hemoglobin. Mildronate administration resulted in a significant reduction of the circulating pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α and a trend toward reduction of IL-6, and IL-17 in SCD mice. We found higher level of hypoxia in the medulla of SCD mice compared to control mice. Mildronate injections did not reduced hypoxia in renal medulla of SCD mice. Also, mildronate injections did not change MDA levels in plasma or kidney of SCD mice. We found a significant reduction of proteinuria in SCD mice injected with mildronate compared to SCD mice injected with vehicle. Analysis of H&E-stained kidneys showed significant reduction of glomerular size and medullar congestion areas in the mildronate injected SCD mice. Urinary KIM-1 levels were reduced by mildronate administration. CONCLUSIONS: Our results demonstrate that mildronate ameliorates glomerular and tubular injury in SCD mice and reduces capillary congestion. Mildronate improves renal function, in part by reducing inflammation but not through the reduction of hypoxia or oxidative stress. In future, we plan to assess the mildronate’s impact on kidney function and vessel inflammation. This work was supported by NIH grants R01HL125005, U54MD007597, and SC1HL150685. We thank Xiaomei Niu for helping with the Bio-Plex assay. 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.

Inhibition of NF-κB Activation Improves Non-Nitric Oxide-Mediated Cutaneous Microvascular Function in Reproductive-Aged Healthy Women

PURPOSE: The human cutaneous circulation is used as an in vivo bioassay to examine mechanisms of systemic vascular function and dysfunction in health and disease. In cohorts of patients with accelerated cardiovascular disease risk, systemic interventions to investigate inflammatory mechanisms [e.g., oral salsalate; nuclear factor kappa-B (NF-κB) inhibitor] have been used to elucidate mechanisms underlying microvascular dysfunction. In healthy control subjects, inhibition of inflammatory mechanisms modestly reduces microvascular function. We hypothesize that inhibition of NF-κB activation may impair nitric oxide (NO)-mediated cutaneous microvascular function in reproductive-aged healthy women. METHODS: In a randomized, single-blind, placebo-controlled design, oral salsalate (1500mg/b.i.d./5 days) was used to assess the impact of systemic inflammation on cutaneous microvascular function. Two intradermal microdialysis fibers were placed into the ventral forearm skin of eleven healthy women [Mean (SEM): 34 (2) yrs]. Laser-Doppler flowmetry was used to measure cutaneous blood flow while increasing concentrations of acetylcholine (10−10 to 10−1 M; endothelium-dependent vasodilator) were perfused with a control (lactated Ringer’s) or a NO synthase inhibitor (15 mM NG-nitro-L-arginine methyl ester). Following the dose-response protocol, maximal cutaneous vasodilation was induced by increasing local skin temperature to 43°C and perfusing 28 mM sodium nitroprusside. Cutaneous vascular conductance (CVC) was calculated as the quotient of red blood cell flux over mean arterial pressure and normalized as a percentage of the site-specific maximum (%CVCmax). The NO-dependent contribution was calculated as the difference between the area under the dose-response curve (AUC) of the control and the NO synthase-inhibited sites. RESULTS: There was an overall main effect of salsalate treatment ( p < 0.001). Both the control and NO synthase-inhibited sites shifted upwards following salsalate treatment (Placebo - Control: 45.78 (8.81)% vs NO synthase inhibited: 33.62 (8.10)%; Salsalate - Control: 56.30 (8.48)% vs NO synthase inhibited: 41.57 (7.85)%; both p < 0.05). There was no change in the NO-dependent contribution between treatments (Placebo: 164.30 (35.40) AU vs Salsalate: 180.30 (51.17) AU; p=0.7028).CONCLUSION: Inhibition of NF-κB activation improved cutaneous microvascular function in reproductive-aged healthy women. This improvement was mediated via non-nitric oxide-dependent mechanisms. Supported by NIH Grant R01 HL161000 (LMA) and Diversity Supplement R01 HL161000-02S2 (VGC). 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.

NFAT5 upregulates osmolyte transporters to protect IMCD3 cells against hyperosmolarity

The kidney inner medulla is one of the only places in the body exposed to extremely high osmolarity, reaching up to 1300 mOsm, while the iso-osmotic cortex and plasma are ≍300m mOsm. Renal epithelial cells need a mechanism to address the stress that comes with hyperosmolarity. One possible mechanism is regulation of osmolyte transporters to increase intracellular osmolyte concentration and reduce osmotic stress. NFAT5 is an important factor in regulating renal epithelial cell responses to high osmolarity. We hypothesize that NFAT5 is protective in inner medullary collecting duct cells (IMCD3) via upregulation of key transporters. To test this hypothesis, we used CRISPR to delete NFAT5 in IMCD3 cells. IMCD3 -NFAT5 null (NFAT5 null) and control (Ctl, IMCD3) cells were used and cell death assessed. Cells were plated at the same concentrations, in media (320mmol) with additional osmolarities of: urea (160mmol), mannitol (160mmol) and salt (40 and 80 mmol). Cell death was measured over 5 days using Janus Green B, which permeates living cell membranes. Cells were incubated with Janus Green B, lysed and then absorbance measured with a spectrophotometer to determine concentration. Data were analyzed as mean ± SEM; cell confluence, two-way ANOVA, n=3-5. Total mRNA was also isolated for qPCR analysis and data expressed as a fold change (DCT) over housekeeping (PPIA). At iso-osmolarity (»300mOsm), no differences were observed between NFAT5 null and Ctl cells. However, by day 3, NFAT5 null cells had reduced cell growth in all hyperosmolar conditions: urea 160 mmol (1.192 ± 0.225 vs. 1.953 ± 0.167 Wt ****p<0.0001, n=4), mannitol 160 mmol (0.852 ± 0.135 vs. 1.683 ± 0.283 Wt ****p<0.0001, n=3-4), NaCl 40 mmol (1.745 ± 0.210 vs. 2.020 ± 0.252 Wt ****p<0.0001, n=4-5), NaCl 80 mmol (0.385 ± 0.059 vs. 1.089 ± 0.191 Wt ****p<0.0001, n=4-5). Interestingly, NFAT5 null cells recovered growth patterns similar to Ctl cells by day 5. This suggests that although NFAT5 is essential in the cellular response hyperosmolarity, other mechanisms are present that allow for adaptation over time. To address a possible mechanism, we performed qPCR investigating common osmo-protective genes. We hypothesized that expression is lower in the NFAT5 null cells as compared to Wt. When cultured with 40 mmol of NaCl (n=2), we found that the NFAT5 null cells showed a 3.31 fold reduction for SMIT1, a 2924.98 fold reduction for TauT, and a 4.91 fold reduction for BGT1 as compared to Wt, each of which are osmolyte transporters. Additionally, we saw a 161.46 fold reduction for AQP1, a water channel and a 4.91 fold reduction for Akr1b3, which influences water homeostasis, as compared to Wt. Together, these results suggest that NFAT5 is a critical regulator of both osmolyte and transporters. Here, we show that loss of NFAT5 reduces IMCD cell viability in the face of hyperosmotic stress, possibly through a reduction in key osmolyte transporters. These data suggest that NFAT5 is key in the renal epithelial cell response to, and survival in, the hyperosmolar environment of the inner medulla. Undergraduate Research Opportunity Program grant NIH NIDDK DK115660-01A1 University of Utah DOIM, UCares. 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.

PNPLA3 I148M Promotes Fatty Liver Disease While Preserving Cardiac Health

Metabolic-associated fatty liver disease (MAFLD) and cardiovascular disease (CVD) are commonly associated comorbidities. A common genetic variant in the PNPLA3 gene, rs738409 (I148M), is the greatest genetic risk factor for development and progression of MAFLD; however, MAFLD patients expressing PNPLA3 I148M display modest protection against coronary artery disease (CAD) and adverse cardiovascular events relative to wildtype carriers. Currently, the exact mechanisms by which PNPLA3 I148M causes MAFLD and preserves cardiac health are not well understood. Previous work suggests that the variant increases susceptibility to fatty liver disease by altering hepatic lipid metabolism, which may explain a potential mechanism for its cardioprotective effects. In order to investigate how PNPLA3 I148M might increase susceptibility to MAFLD while reducing risk of CVD we expressed either human wildtype (WT) PNPLA3 or PNPLA3 I148M as well as a GFP control specifically in the livers of PNPLA3 knockout mice. Our data demonstrate that hepatic expression of PNPLA3 I148M reduces secretion of VLDL TAGs from adipose tissue derived fatty acids. In agreement with prior investigations, mice expressing PNPLA3 I148M and fed a chow diet demonstrated significant increases in hepatic accumulation of neutral lipids (triglycerides & cholesterol esters) relative to WT and GFP expressing mice. Similarly, when fed a fatty liver promoting diet (D009100310) high in fructose, cholesterol, and saturated fats there remained a strong trend for greater liver to body weight ratios and hepatic TAGs in mice expressing the variant when compared to WT expressing mice (p=0.052 and p=0.071 respectively). Changes in hepatic lipid accumulation were not due to any alterations in insulin sensitivity, daily food consumption, energy expenditure or oxygen consumption between WT and I148M groups. Echocardiography revealed that hearts of mice expressing WT PNPLA3 had increases in left ventricular thickness (p=0.051) and left ventricle weights following dietary treatment that was not apparent in the I148M expressing mice. Moreover, there was a reduction in E/A ratios, a sensitive measure of diastolic functioning, in GFP and WT expressing mice post-treatment, that was not seen in the I148M mice. Overall, these findings indicate that PNPLA3 I148M promotes fatty liver, yet protects against diet-induced effects on ventricular remodeling and diastolic functioning. Our next steps will be to assess measures of atherosclerotic plaque formation around major blood vessels and investigate the metabolic pathways altered by PNPLA3 I148M. NIH R00 DK114471 NIH R01 DK126743 USDA grant no. 2021-09425 Pilot feasibility grant from University of Michigan: P30DK020572 Detroit Cardiovascular Training grant NIH 2T32HL120822. 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.

Endogenous retroelement expression in the gut microenvironment of people living with HIV-1

Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC≤-1 or≥1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).

Targeting disparate spaces: new technology and old tools.

A growing number of inexpensive, publicly available, validated air quality monitors are currently generating granular and longitudinal data on air quality. The expansion of interconnected networks of these monitors providing open access to longitudinal data represents a valuable data source for health researchers, citizen scientists, and community members; however, the distribution of these data collection systems will determine the groups that will benefit from them. Expansion of these and other exposure measurement networks represents a unique opportunity to address persistent inequities across racial, ethnic, and class lines, if the distribution of these devices is equitable. We present a lean template for local implementation, centered on groups known to experience excess burden of pulmonary disease, leveraging five resources, (a) publicly available, inexpensive air quality monitors connected via Wi-Fi to a centralized system, (b) discharge data from a state hospital repository (c) the U.S. Census, (d) monitoring locations generously donated by community organizations and (e) NIH grant funds. We describe our novel approach to targeting air-quality mediated pulmonary health disparities, review logistical and analytic challenges encountered, and present preliminary data that aligns with a growing body of research: in a high-burden zip code in Durham North Carolina, the census tract with the highest proportions of African Americans experienced worse air quality than a majority European-American census tract in the same zip code. These results, while not appropriate for use in causal inference, demonstrate the potential of equitably distributed, interconnected air quality sensors.

Characterization of CD4+ t cell subsets during Chlamydia muridarum genital infection

Characterization of CD4+ T cell subsets in a stressed mouse model during Chlamydia muridarum genital infection. Katrina Davis, Tesfaye Belay. Department of Applied Sciences and Mathematics. Chlamydia genital infection caused by Chlamydia trachomatis is highly prevalent in low socioeconomic status populations. Whether stress is a risk factor for the high incidence of chlamydia remains to be investigated. CD4+ T cells are the major immune cells that fight chlamydia genital infection; however, the dynamics of CD4+ T cells during stressful conditions and chlamydia infection are unclear. The profiles of CD4+ T cell signature cytokines: T helper 1 (IL-12, Interferon-gamma (IFN-γ), Thelper2 (IL-4, IL-5, IL-13), Thelper17(IL-17), and Treg (IL-10). Mice were stressed for 5 minutes daily for 21 days and infected with 1x 105 IFU/ml of Chlamydia muridarum intravaginally. Non-stressed mice were infected as controls. Mice were sacrificed 7 days post-infection, the spleen and lymph nodes were harvested, and T cells were purified and proliferated for 72 h. Determination of the levels of cytokines in culture supernatants of T cells from stressed and non-stressed mice are underway for poster presentation. Supported by the NIH grant # 1R15AI124156-01 awarded to BSU

Combinatorial effects of resazomycins and azithromycin against Neisseria gonorrhoeae

Gonorrhea is a sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae. The WHO estimates 87 million new gonococcal infections occur each year making it the second most common sexually transmitted bacterial infection. When left untreated, gonorrhea can cause pelvic inflammatory disease and infertility in women. Treatment is further complicated by the fact that N. gonorrhoeae has acquired resistance to all antibiotics approved for treatment including the gold standard ceftriaxone. One way to address the growing problem of antibiotic resistance is to administer drugs in combination in the hope the two are more effective together than alone. Resazomycins, a novel family of resazurin (Rz) based antibiotics, exhibit antimicrobial activity against N. gonorrhoeae and Francisella tularensis. Here, we used E-test assays to determine if there was a synergistic bactericidal effect between resazomycins and azithromycin, which is commonly used to treat gonorrhea. Multiple clinical isolates of N. gonorrhoeae were more sensitive to azithromycin in the presence of Rz. However, additional testing in a checkerboard assay revealed no synergistic effect between azithromycin and Rz or another resazomycin, resorufin pentyl ether (RPE). Upon further investigation, we determined the conflicting results between the E-test and checkerboard method were due to differences in bacterial load. When the E-test assays were repeated using a lower inoculum of N. gonorrhoeae, increased sensitivity to azithromycin was not observed in the presence of Rz. In the future, we plan to test resazomycins in combination with other antibiotics against N. gonorrhoeae, particularly extensively drug-resistant strains. (This research was made possible by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and the NASA West Virginia Space Grant Consortium, Grant # 80NSSC20M0055)

Role of LpnA in Francisella tularensis Susceptibility to Resazurin

Francisella tularensis is classified by the Centers for Disease Control and Prevention as a Category A bioterrorism agent due to its low infectious dose, ease of aerosolization, and high mortality rate. Given there is no licensed tularemia vaccine and the possibility of antibiotic-resistant F. tularensis strains, there is an urgent need to develop new treatments against this bacterium. Resazurin (Rz) has bactericidal activity against F. tularensis and other gram-negative bacteria including the human pathogens Neisseria gonorrhoeae and Helicobacter pylori. However, the mechanism of action for Rz has yet to be determined. To identify targets of Rz, we screened for Rz-resistant (Rzr) mutants of F. tularensis LVS. Whole genome sequencing was performed on 42 Rzr isolates and all contained mutations within the coding regions of FTL_0421 and FTL_1504. FTL_0421 encodes for LpnA, the major lipoprotein in F. tularensis. The FTL_0421 mutation in Rzr1 resulted in loss of LpnA expression which correlated with increased sensitivity to osmotic stress. To investigate the individual role of lpnA in Rz-resistance, a deletion mutant (ΔlpnA) was generated using standard molecular genetics techniques. An agar dilution assay was then performed to determine the Rz susceptibility of the ΔlpnA strain. A minimum inhibitory concentration (MIC) of 2.75μg/mL was achieved for the ΔlpnA and the LVS wild-type strain. This data suggests mutation of lpnA alone is not sufficient to confer resistance to resazurin. In the future, we will investigate whether lpnA in combination with other genes like FTL_1504 play a role in resazurin susceptibility in F. tularensis. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence &amp; the NASA Space Grant Consortium)

Utilization of Double-Immunofluorescence Microscopy to determine the role of FTL_1199 in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is an intracellular bacterium that is the causative agent of the zoonotic disease, tularemia. F. tularensis invades host erythrocytes during infection, a phenomenon that leads to increased colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, an RNA-seq analysis was conducted to identify genes whose transcription was affected by the presence of erythrocytes. A putative transcriptional regulator, FTL_1199 was determined to be modulated in the presence of erythrocytes. We hypothesized that this gene may therefore play a role in erythrocyte invasion. Deletion of FTL_1199 resulted in a significant reduction in the invasion of rabbit erythrocytes, and complementation of FTL_1199 restored this strain’s ability to acquire the cytoplasmic space of erythrocytes. These findings were determined by a gentamicin protection assay to quantify erythrocyte invasion. To validate these findings, we conducted double-immunofluorescence microscopy. F. tularensis strains incubated with rabbit erythrocytes to allow for invasion. Cells were probed with anti-F. tularensis antibodies followed by red fluorescent secondary antibodies. Cells were washed and erythrocytes were permeabilized. Bacteria were probed a second time utilizing a green fluorescent secondary antibody. Therefore, intracellular bacteria were only labeled with a singular secondary antibody, while extracellular bacteria were tagged with two secondary antibodies. Intracellular bacterial enumeration showed that deletion of FTL_1199 resulted in a significant reduction of invasion (p = 0.0253), while FTL_1199 complementation to the deletion mutant restored invasion to that of wild type (p = 0.3258). These data demonstrate that FTL_1199 plays an important role in erythrocyte invasion. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).

Lipidomic analysis by LC-MS/MS of wildtype, mutant and VBNC strains of Francisella tularensis LVS.

Francisella tularensis is one of the many bacteria that can enter a viable but nonculturable state (VBNC). The VBNC state acts as an adaptive strategy that allows for long-term survival of bacteria in unfavorable environments. A defining characteristic of VBNC bacteria is a loss of culturability, making VBNC bacteria difficult to detect in the environment using standard plating assays. Failure to identify VBNC bacteria in the food industry or water distribution centers poses a serious risk to human health. Our preliminary data suggests that lipid metabolism plays a role in VBNC adaptation in F. tularensis. To investigate further, genetic deletion mutants have been constructed from two predicted lipid metabolism genes, FTL_1569 (phosphoglycolate phosphatase) and FTL_1570 (phospholipase D family protein). Lipid extracts were collected from wildtype, mutant and VBNC strains of F. tularensis to determine any lipid species associated with the VBNC state. A single-phase lipid extraction method was used to obtain lipids from F. tularensis cultures grown for 24 hours (culturable) and 336 hours (VBNC). Liquid chromatography with tandem mass spectrometry (LC-MS-MS) and the ThermoFisher Compound Discoverer software were utilized for lipid identification, and comparative analyses. Differences in the lipid profile of wildtype, mutant and VBNC strains of F. tularensis have been identified. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Abstract LB377: The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice

Abstract Head and neck cancer (HNC), commonly known as oral cancer, is a devastating disease and the 6th most common cancer worldwide. Most HNC patients are diagnosed with advanced-stage disease for which the 5-year survival is below 50%, stressing the need for chemoprevention. Recently, we have reported that the combination of resveratrol and EGCG induces synergistic apoptosis and inhibits xenografted HNC growth by inhibiting the AKT-mTOR pathway. This study investigated the chemopreventive efficacy of resveratrol, EGCG, and their combination using the 4NQO-induced oral carcinogenesis model. C57BL/6 mice were exposed to 4-NQO (50 μg/ml) via drinking water for 10 weeks, followed by treatment with vehicle (50% sweetened condensed milk), resveratrol (30 mg/kg), EGCG (30 mg/kg) and their combination for 8 weeks, 5 days/week. The mice were sacrificed on week 24, and the number of visible and microscopic lesions was counted. Resveratrol alone and in combination with EGCG significantly inhibited the number of visible lesions. In contrast, the number of microscopic lesions and lesion area was significantly inhibited only in the combination group. The expression of Ki-67 was also significantly inhibited in resveratrol and combination groups when compared with untreated control. Furthermore, RNASeq and qPCR analysis using an HNC cell line identified GDF15, ATF3, p21, p27, and Bim as significantly upregulated genes, with GDF15 being the most upregulated one. Expression of GDF15 and ATF3 proteins was confirmed by Western blotting. Taken together, our data strongly demonstrate the chemopreventive potential of the combination of EGCG and resveratrol and pave the way for further clinical developments. (Supported by NIH Grants R15DE032063 and P20GM103434) Citation Format: Adeoluwa A. Adeluola, Lukmon Raji, Saroj Sigdel, ASM Anisuzzaman, A.R.M. Ruhul Amin. The combination of epigallocatechin gallate (EGCG) and resveratrol prevents 4-NQO-induced oral carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB377.

Spectrum of prevalent cardiovascular diseases in urban Port-au-Prince, Haiti: a population-based cross-sectional study

Eighty percent of global cardiovascular disease (CVD) is projected to occur in low- and middle -income countries (LMICs), yet local epidemiological data are scarce. We provide the first population-based, adjudicated CVD prevalence estimates in Port-au-Prince, Haiti to describe the spectrum of heart disease and investigate associated risk factors. Demographic, medical history, clinical, imaging and laboratory data were collected among adults recruited using multistage random sampling from 2019 to 2021. Prevalent CVD (heart failure, stroke, ischemic disease) were adjudicated using epidemiological criteria similar to international cohorts. Multivariable Poisson regressions assessed relationships between risk factors and prevalent CVD. Among 3003 participants, median age was 40 years, 58.1% were female, 70.2% reported income <1 USD/day, and all identified as Black Haitian. CVD age-adjusted prevalence was 14.7% (95% CI 13.3%, 16.5%), including heart failure (11.9% [95% CI 10.5%, 13.5%]), stroke (2.4% [95% CI 1.9%, 3.3%]), angina (2.1% [95% CI 1.6%, 2.9%]), myocardial infarction (1.0% [95% CI 0.6%, 1.8%]), and transient ischemic attack (0.4% [95% CI 0.2%, 1.0%]). Among participants with heart failure, median age was 57 years and 68.5% of cases were among women. The most common subtype was heart failure with preserved ejection fraction (80.4%). Heart failure was associated with hypertension, obesity, chronic kidney disease, depression, and stress. Early-onset heart failure prevalence is alarmingly high in urban Haiti and challenge modelling assumptions that ischemic heart disease and stroke dominate CVDs in LMICs. These data underscore the importance of local population-based epidemiologic data within LMICs to expedite the selection and implementation of evidence-based cardiovascular health policies targeting each country's spectrum of heart disease. This study was funded by NIH grants R01HL143788, D43TW011972, and K24HL163393, clinicaltrials.govNCT03892265.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization

Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

335 Federal Research Funding Trends for Cerebrovascular Neurosurgeons: The Decline of the Cerebrovascular Surgeon-Scientist?

INTRODUCTION: With the rise of endovascular neurosurgery, the number of cerebrovascular (CV) surgeons has grown. However, it is unclear whether the number of CV surgeon-scientists has concomitantly increased. METHODS: Publicly available data were collected on currently practicing academic CV surgeons in the US. Inflation-adjusted NIH funding between 2009-2021 was surveyed using NIH RePORTER. The K12 Neurosurgeon Career Development Program grant data were queried for CV-focused grants. Pearson R correlation was used for statistical analysis. RESULTS: From 2009-2021, NIH funding has increased: in total (p = 0.0318), to neurosurgeons (p &lt; 0.0001), to CV research projects (p &lt; 0.0001), and to CV surgeons (p = 0.0018). There has been an increase in the total number of CV surgeons (p &lt; 0.0001), NIH-funded CV surgeons (p = 0.0034), and the percent of CV surgeons with NIH funding (p = 0.370). Additionally, active NIH grant dollars per CV surgeon (p = 0.0047) and number of NIH grants per CV surgeon (p = 0.0078) has increased. However, the percent of neurosurgeon-awarded NIH grants awarded specifically to CV surgeons has trended down over the past decade (p=0.3664). Analysis of early-career research grants showed a decline in the proportion of K12 applicants (p = 0.1840). CONCLUSIONS: Our data show that there are more CV surgeons than ever, and there has been a proportional increase in number of NIH-funded CV surgeons. However, there are fewer early-career neurosurgeons pursuing CV-focused projects, and the proportion of neurosurgeon-awarded NIH grants given to CV surgeons has trended down. These findings suggest a declining trend in CV surgeons pursuing research careers.

Elements of successful NIH grant applications

Is there a formula for a competitive NIH grant application? The Serenity Prayer may provide one: "Grant me the serenity to accept the things I cannot change, the ability to change the things I can, and the wisdom to know the difference." But how to tell the difference? In this Perspective, we provide an inclusive roadmap—elements of NIH funding. Collectively, we have over 30 y of peer review experience as NIH Scientific Review Officers in addition to over 30 y of program experience as NIH Program Officers. This article distills our NIH experience. We use Euclid’s 13-book landmark, The Elements, as our template to humbly share what we learned. We have three specific aims: inform, guide, and motivate prospective applicants. We also address ways that support diversity and inclusion among applicants and young investigators in biomedical research. The elements we describe come from a wide range of sources. Some themes will be general. Some will be specific. All will be candid. The ultimate goal is a competitive application, serenity, and hopefully both.

Deconstructing pathological tau by biological process in early stages of Alzheimer disease: a method for quantifying tau spatial spread in neuroimaging

Neuroimaging studies often quantify tau burden in standardized brain regions to assess Alzheimer disease (AD) progression. However, this method ignores another key biological process in which tau spreads to additional brain regions. We have developed a metric for calculating the extent tau pathology has spread throughout the brain and evaluate the relationship between this metric and tau burden across early stages of AD. 445 cross-sectional participants (aged≥50) who had MRI, amyloid PET, tau PET, and clinical testing were separated into disease-stage groups based on amyloid positivity and cognitive status (older cognitively normal control, preclinical AD, and symptomatic AD). Tau burden and tau spatial spread were calculated for all participants. We found both tau metrics significantly elevated across increasing disease stages (p<0.0001) and as a function of increasing amyloid burden for participants with preclinical (p<0.0001, p=0.0056) and symptomatic (p=0.010, p=0.0021) AD. An interaction was found between tau burden and tau spatial spread when predicting amyloid burden (p=0.00013). Analyses of slope between tau metrics demonstrated more spread than burden in preclinical AD (β=0.59), but then tau burden elevated relative to spread (β=0.42) once participants had symptomatic AD, when the tau metrics became highly correlated (R=0.83). Tau burden and tau spatial spread are both strong biomarkers for early AD but provide unique information, particularly at the preclinical stage. Tau spatial spread may demonstrate earlier changes than tau burden which could have broad impact in clinical trial design. This research was supported by the Knight Alzheimer Disease Research Center (Knight ADRC, NIH grants P30AG066444, P01AG026276, P01AG003991), Dominantly Inherited Alzheimer Network (DIAN, NIH grants U01AG042791, U19AG03243808, R01AG052550-01A1, R01AG05255003), and the Barnes-Jewish Hospital Foundation Willman Scholar Fund.

Abstract 748: Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial

Abstract Introduction: Higher consumption of dietary sulforaphane (SFN), a phytochemical found in many cruciferous vegetables, has been shown to be associated with lower risk of lung cancer (LC). SF activates the NRF2-pathway, which initiates cellular detoxification, antioxidant, and anti-inflammatory responses. To date, no chemoprevention trial for LC has examined the impact of SFN on LC-related gene expression (GE) as an endpoint in nasal and bronchial tissue. The objective of our study was to examine whether oral intake of SF for 12 months impacts the GE of previously identified LC- and premalignant lesions (PML)-related genes in bronchial and nasal brushing samples. Methods: A randomized, placebo-controlled clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in Western Pennsylvania, USA. Forty-three subjects were randomly assigned to the SFN treatment (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Nasal swabs and bronchial brushings were obtained at baseline and after 12 months. Among the 37 participants who completed the study, RNA was extracted from their nasal swabs and bronchial brushings. We constructed single sample gene signature scores (GSS) using the GSVA algorithm for the following: 1) the NRF2-pathway, 2) genes associated with LC risk in bronchial biopsies, and 3) genes associated with PML. The genes in these sets were stratified by their up- or down-regulation in LC or PML. We evaluated the association between the interaction of SFN supplementation over time (pre-/post-treatment) on GSS using linear mixed models, adjusted for the fixed effects of age at baseline, RNA integrity number (RIN), packyears of smoking, and random effect of participant. Results: GE data was available for 31 participant pairs with bronchial and/or nasal samples. Within nasal tissue, SFN treatment increased both the GSSs of the NRF2-pathway (p=0.0089) and the downregulated LC-risk genes (p=0.0047), and decreased the upregulated LC-risk genes (p=0.012) over time, whereas SFN had no significant impact on GSSs of the NRF2-pathway and LC-risk genes in the bronchial tissue. No association between SFN treatment over time and the PML-related GSS was observed in nasal or bronchial tissue. Conclusion: Our initial analysis indicates that SFN treatment over 12 months impacted LC-associated GE in nasal tissue but not in bronchial tissue from former smokers. The discrepancy results may be due to small sample size and additional studies are warranted to confirm if SFN has direct impact on LC-risk related genes in bronchial tissue (NIH grant No. R01CA213123). Citation Format: Kathryn Demanelis, Avrum Spira, Yohana Kefella, Renwei Wang, Jennifer Adams-Haduch, Thomas W. Kensler, Jennifer E. Beane, David O. Wilson, Jian-Min Yuan. Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 748.

Abstract 7235: Exosome-mediated delivery of siRNA against NRF2 for treatment of lung cancer

Abstract Nuclear factor erythroid 2-related factor 2 (NRF2) activation is the third most common genetic event in lung adenocarcinomas (25%), primarily induced by loss of function mutations in its master regulator, KEAP1. NRF2 is a key modulator of tumor growth and metabolic reprogramming, resulting in resistance to traditional chemotherapeutics. Due to its role in modulating oxidative stress, sustained NRF2 overexpression promotes disease progression, particularly in lung cancer. This study demonstrates the effective knockdown of NRF2 expression by small interfering RNA (siRNA) delivered via a nanoplatform based on bovine colostrum exosomes to lung cancer cells, both in vitro and in vivo, resulting in inhibition of proliferation and disease progression. Bovine colostrum is an abundant source of exosomes. Here we present a novel exosome-polyethyleneimine matrix (EPM) to deliver therapeutic siRNA targeting NRF2 expression (siNRF2). Specific delivery of the EPM to tumors was further enhanced via folic acid (FA) functionalization, targeting folate receptors highly expressed on lung tumor cells. Exosomes were isolated from standardized colostrum powder through rehydration and differential centrifugation, and characterized by size, polydispersity index (pdi) and surface charge by Zetasizer, and the presence of surface protein markers by Western blot. siNRF2 was loaded onto the EPM, and the resultant EPM-siNRF2 complex was harvested by PEG precipitation. Entrapment efficiency was determined using 5’-32P-labeled siRNA as a tracer. The gene knockdown efficiency of EPM-siNRF2 was assessed in lung cancer (A549) cells by Western blot and the therapeutic effect of target knockdown in vitro was confirmed by analyzing the corresponding modulation in downstream targets of NRF2. The anti-tumor effect was then evaluated using both subcutaneous and orthotropic lung tumor models in immunocompromised mice. The EPM exhibited high entrapment efficiency (&amp;gt;90%) of siNRF2 (up to 20 μg) and protected entrapped siRNA from enzymatic degradation upon exposure to RNases. EPM-siNRF2 dose dependently decreased NRF2 expression in A549 lung cancer cells. When formulated with FA-functionalized exosomes, EPM-siNRF2 inhibited subcutaneous and orthotropic lung tumors in mice by over 57% (p&amp;lt;0.01) and 84% (p&amp;lt;0.001), respectively. Mice treated with FA-EPM-siNRF2 showed decreased levels of NRF2 in tumor tissue, but not in the adjacent lung, indicating FA-mediated targeted delivery. This bovine colostrum exosome based EPM technology showcases high efficiency in gene knockdown and lung tumor inhibition, providing a promising nanoplatform for delivering nucleic acid therapeutics to the tumor site. Funding: Supported from the NIH grant R44 CA-221487, 3P Biotechnology Contract and, in part, Agnes Brown Duggan Endowment. Keywords: Bovine colostrum, Exosomes, NRF2, siRNA, Lung cancer. Citation Format: Raghuram Kandimalla, Margaret Wallen, Jeyaprakash Jeyabalan, Disha N. Moholkar, Wendy Spencer, Ramesh C. Gupta, Farrukh Aqil. Exosome-mediated delivery of siRNA against NRF2 for treatment of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7235.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5564.