Inflammatory proteins are associated with mortality in a middle-aged diverse cohort.

BACKGROUND: Recent data indicate a decline in overall longevity in the United States. Even prior to the COVID-19 pandemic, an increase in midlife mortality rates had been reported. Life expectancy disparities have persisted in the United States for racial and ethnic groups and for individuals living at low socioeconomic status. These continued trends in mortality indicate the importance of examining biomarkers of mortality at midlife in at-risk populations. Circulating levels of cytokines and inflammatory markers reflect systemic chronic inflammation, which is a well-known driver of many age-related diseases. METHODS: In this study, we examined the relationship of nine different inflammatory proteins with mortality in a middle-aged socioeconomically diverse cohort of African-American and White men and women (n = 1122; mean age = 47.8 years). RESULTS: We found significant differences in inflammatory-related protein serum levels between African-American and White middle-aged adults. E-selectin and fibrinogen were significantly higher in African-American adults. IFN-γ, TNF-α trimer, monocyte chemoattractant protein-1 (MCP-1), soluble receptor for advanced glycation end-products (sRAGE) and P-selectin were significantly higher in White participants compared to African-American participants. Higher levels of E-selectin, MCP-1 and P-selectin were associated with a higher mortality risk. Furthermore, there was a significant interaction between sex and IL-6 with mortality. IL-6 levels were associated with an increased risk of mortality, an association that was significantly greater in women than men. In addition, White participants with high levels of sRAGE had significantly higher survival probability than White participants with low levels of sRAGE, while African-American participants had similar survival probabilities across sRAGE levels. CONCLUSIONS: These results suggest that circulating inflammatory markers can be utilized as indicators of midlife mortality risk in a socioeconomically diverse cohort of African-American and White individuals.

Association of extracellular vesicle inflammatory proteins and mortality.

Even before the COVID-19 pandemic declines in life expectancy in the United States were attributed to increased mortality rates in midlife adults across racial and ethnic groups, indicating a need for markers to identify individuals at risk for early mortality. Extracellular vesicles (EVs) are small, lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids. Given their role as intercellular communicators and potential biomarkers of disease, we explored whether circulating EVs may be markers of mortality in a prospective, racially, and socioeconomically diverse middle-aged cohort. We isolated plasma EVs from 76 individuals (mean age = 59.6 years) who died within a 5 year period and 76 surviving individuals matched by age, race, and poverty status. There were no significant differences in EV concentration, size, or EV-associated mitochondrial DNA levels associated with mortality. We found that several EV-associated inflammatory proteins including CCL23, CSF-1, CXCL9, GDNF, MCP-1, STAMBP, and 4E-BP1 were significantly associated with mortality. IL-10RB and CDCP1 were more likely to be present in plasma EVs from deceased individuals than in their alive counterparts. We also report differences in EV-associated inflammatory proteins with poverty status, race, and sex. Our results suggest that plasma EV-associated inflammatory proteins are promising potential clinical biomarkers of mortality.

The accelerated aging phenotype: The role of race and social determinants of health on aging.

The pursuit to discover the fundamental biology and mechanisms of aging within the context of the physical and social environment is critical to designing interventions to prevent and treat its complex phenotypes. Aging research is critically linked to understanding health disparities because these inequities shape minority aging, which may proceed on a different trajectory than the overall population. Health disparities are characteristically seen in commonly occurring age-associated diseases such as cardiovascular and cerebrovascular disease as well as diabetes mellitus and cancer. The early appearance and increased severity of age-associated disease among African American and low socioeconomic status (SES) individuals suggests that the factors contributing to the emergence of health disparities may also induce a phenotype of 'premature aging' or 'accelerated aging' or 'weathering'. In marginalized and low SES populations with high rates of early onset age-associated disease the interaction of biologic, psychosocial, socioeconomic and environmental factors may result in a phenotype of accelerated aging biologically similar to premature aging syndromes with increased susceptibility to oxidative stress, premature accumulation of oxidative DNA damage, defects in DNA repair and higher levels of biomarkers of oxidative stress and inflammation. Health disparities, therefore, may be the end product of this complex interaction in populations at high risk. This review will examine the factors that drive both health disparities and the accelerated aging phenotype that ultimately contributes to premature mortality.

Low-Level Ionizing Radiation Induces Selective Killing of HIV-1-Infected Cells with Reversal of Cytokine Induction Using mTOR Inhibitors.

HIV-1 infects 39.5 million people worldwide, and cART is effective in preventing viral spread by reducing HIV-1 plasma viral loads to undetectable levels. However, viral reservoirs persist by mechanisms, including the inhibition of autophagy by HIV-1 proteins (i.e., Nef and Tat). HIV-1 reservoirs can be targeted by the "shock and kill" strategy, which utilizes latency-reversing agents (LRAs) to activate latent proviruses and immunotarget the virus-producing cells. Yet, limitations include reduced LRA permeability across anatomical barriers and immune hyper-activation. Ionizing radiation (IR) induces effective viral activation across anatomical barriers. Like other LRAs, IR may cause inflammation and modulate the secretion of extracellular vesicles (EVs). We and others have shown that cells may secrete cytokines and viral proteins in EVs and, therefore, LRAs may contribute to inflammatory EVs. In the present study, we mitigated the effects of IR-induced inflammatory EVs (i.e., TNF-α), through the use of mTOR inhibitors (mTORi; Rapamycin and INK128). Further, mTORi were found to enhance the selective killing of HIV-1-infected myeloid and T-cell reservoirs at the exclusion of uninfected cells, potentially via inhibition of viral transcription/translation and induction of autophagy. Collectively, the proposed regimen using cART, IR, and mTORi presents a novel approach allowing for the targeting of viral reservoirs, prevention of immune hyper-activation, and selectively killing latently infected HIV-1 cells.

Association between GDF15, poverty and mortality in urban middle-aged African American and white adults.

Mortality disparities are influenced by race and poverty. There is limited information about whether poverty influences biologic markers of mortality risk. Emerging data suggests that growth differentiation factor 15 (GDF15) is associated with mortality; however, the interplay between GDF15, sociodemographic factors and mortality is not known. We sought to evaluate the interactions between GDF15 and sex, race and poverty status on mortality. Serum GDF15 was measured in 1036 African American and white middle-aged men and women above and below 125% of the Federal poverty status from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Multivariable adjusted Cox regression models were used to assess the association between log-transformed GDF15 (logGDF15) and 12-year mortality outcomes (all-cause, cardiovascular- and cancer-specific outcomes) and interactions with sex, race and poverty status. Likelihood ratio tests were used to assess significance of the interaction terms. Median GDF15 was 655.2 pg/mL (IQR = 575.1). During 12.2 years of follow-up, 331 died of which 94 cardiovascular- and 87 were cancer-specific deaths. One unit of increase in logGDF15 was associated with a hazard ratio for all-cause mortality, cardiovascular- and cancer-specific mortality of 2.26 (95% confidence interval [CI], 1.94-2.64), 2.74 (95%CI, 2.06-3.63) and 1.41 (95%CI, 1.00-2.00), respectively. There was an interaction between logGDF15 and poverty status on all-cause mortality (p<0.05). The GDF15×poverty status interaction term improved model calibration for all-cause mortality. Our study provides the first evidence that the effect of elevated GDF15 on all-cause mortality is modified by poverty status.

Extracellular vesicles in diabetes mellitus induce alterations in endothelial cell morphology and migration.

BACKGROUND: Inflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. Extracellular vesicles (EVs) are nano-sized particles that contain molecular cargo and circulate in the blood. Here, we examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells. METHODS: We quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort. RESULTS: We found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and ß-cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals. CONCLUSIONS: Higher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease.

Frailty in middle age is associated with frailty status and race-specific changes to the transcriptome.

Frailty is an aging-associated syndrome resulting from diminished capacity to respond to stressors and is a significant risk factor for disability and mortality. Although frailty is usually studied in old age, it is present in mid-life. Given the increases in mortality statistics among middle-aged Americans, understanding molecular drivers of frailty in a younger, diverse cohort may facilitate identifying pathways for early intervention. We analyzed frailty-associated, genome-wide transcriptional changes in middle-aged blacks and whites. Next generation RNA sequencing was completed using total RNA from peripheral blood mononuclear cells (n = 16). We analyzed differential gene expression patterns and completed a parametric analysis of gene set enrichment (PAGE). Differential gene expression was validated using RT-qPCR (n = 52). We identified 5,082 genes differentially expressed with frailty. Frailty altered gene expression patterns and biological pathways differently in blacks and whites, including pathways related to inflammation and immunity. The validation study showed a significant two-way interaction between frailty, race, and expression of the cytokine IL1B and the transcription factor EGR1. The glucose transporter, SLC2A6, the neutrophil receptor, FCGR3B, and the accessory protein, C17orf56, were decreased with frailty. These results suggest that there may be demographic dependent, divergent biological pathways underlying frailty in middle-aged adults.

Loss of RNA-binding protein GRSF1 activates mTOR to elicit a proinflammatory transcriptional program.

The RNA-binding protein GRSF1 (G-rich RNA sequence-binding factor 1) critically maintains mitochondrial homeostasis. Accordingly, loss of GRSF1 impaired mitochondrial respiration and increased the levels of reactive oxygen species (ROS), triggering DNA damage, growth suppression, and a senescent phenotype characterized by elevated production and secretion of interleukin (IL)6. Here, we characterize the pathways that govern IL6 production in response to mitochondrial dysfunction in GRSF1-depleted cells. We report that loss of GRSF1 broadly altered protein expression programs, impairing the function of respiratory complexes I and IV. The rise in oxidative stress led to increased DNA damage and activation of mTOR, which in turn activated NF-κB to induce IL6 gene transcription and orchestrate a pro-inflammatory program. Collectively, our results indicate that GRSF1 helps preserve mitochondrial homeostasis, in turn preventing oxidative DNA damage and the activation of mTOR and NF-κB, and suppressing a transcriptional pro-inflammatory program leading to increased IL6 production.

CRP Stimulates GDF15 Expression in Endothelial Cells through p53.

Growth differentiation factor 15 (GDF15) is a multifunctional, secreted protein that is a direct target gene of p53. GDF15 is a prospective biomarker of cardiovascular disease (CVD). C-reactive protein (CRP), like GDF15, is implicated in inflammation and an independent biomarker of CVD. However, the molecular interactions between GDF15 and CRP remain unexplored. In women, we found a significant relationship between hsCRP and GDF15 serum and mRNA levels. In vitro treatment of cultured human aortic endothelial cells (HAECs) with purified CRP or transfection of a CRP plasmid into HAECs induced GDF15 expression. Dual-luciferase reporter assays confirmed that CRP significantly increased the levels of GDF15 promoter luciferase activity, indicating that CRP induces GDF15 transcription. Chromatin immunoprecipitation (ChIP) assays confirmed that p53 was recruited to both p53 binding sites 1 and 2 in the GDF15 promoter in response to CRP. We have uncovered a linkage between CRP and GDF15, a new clue that could be important in the pathogenesis of endothelial inflammation.

Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes.

Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

Techniques to Induce and Quantify Cellular Senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated ß-galactosidase activity (SA-ß-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including γ-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-ß-gal, γ-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Racial differences in microRNA and gene expression in hypertensive women.

Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.

Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence.

Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease.

Posttranscriptional Regulation of the Inflammatory Marker C-Reactive Protein by the RNA-Binding Protein HuR and MicroRNA 637.

C-reactive protein (CRP), an acute-phase plasma protein, is a major component of inflammatory reactions functioning as a mediator of innate immunity. It has been widely used as a validated clinical biomarker of the inflammatory state in trauma, infection, and age-associated chronic diseases, including cancer and cardiovascular disease (CVD). Despite this, the molecular mechanisms that regulate CRP expression are not well understood. Given that the CRP 3' untranslated region (UTR) is long and AU rich, we hypothesized that CRP may be regulated posttranscriptionally by RNA-binding proteins (RBPs) and by microRNAs. Here, we found that the RBP HuR bound directly to the CRP 3' UTR and affected CRP mRNA levels. Through this interaction, HuR selectively increased CRP mRNA stability and promoted CRP translation. Interestingly, treatment with the age-associated inflammatory cytokine interleukin-6 (IL-6) increased binding of HuR to CRP mRNA, and conversely, HuR was required for IL-6-mediated upregulation of CRP expression. In addition, we identified microRNA 637 (miR-637) as a microRNA that potently inhibited CRP expression in competition with HuR. Taken together, we have uncovered an important posttranscriptional mechanism that modulates the expression of the inflammatory marker CRP, which may be utilized in the development of treatments for inflammatory processes that cause CVD and age-related diseases.

PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity.

Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.

Age-related changes in microRNA levels in serum.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific mRNAs. Altered expression of circulating miRNAs have been associated with age-related diseases including cancer and cardiovascular disease. Although we and others have found an age-dependent decrease in miRNA expression in peripheral blood mononuclear cells (PBMCs), little is known about the role of circulating miRNAs in human aging. Here, we examined miRNA expression in human serum from young (mean age 30 years) and old (mean age 64 years) individuals using next generation sequencing technology and real-time quantitative PCR. Of the miRNAs that we found to be present in serum, three were significantly decreased in 20 older individuals compared to 20 younger individuals: miR-151a-5p, miR-181a-5p and miR-1248. Consistent with our data in humans, these miRNAs are also present at lower levels in the serum of elderly rhesus monkeys. In humans, miR-1248 was found to regulate the expression of mRNAs involved in inflammatory pathways and miR-181a was found to correlate negatively with the pro-inflammatory cytokines IL-6 and TNFα and to correlate positively with the anti-inflammatory cytokines TGFß and IL-10. These results suggest that circulating miRNAs may be a biological marker of aging and could also be important for regulating longevity. Identification of stable miRNA biomarkers in serum could have great potential as a noninvasive diagnostic tool as well as enhance our understanding of physiological changes that occur with age.

Markers of oxidant stress that are clinically relevant in aging and age-related disease.

Despite the long held hypothesis that oxidant stress results in accumulated oxidative damage to cellular macromolecules and subsequently to aging and age-related chronic disease, it has been difficult to consistently define and specifically identify markers of oxidant stress that are consistently and directly linked to age and disease status. Inflammation because it is also linked to oxidant stress, aging, and chronic disease also plays an important role in understanding the clinical implications of oxidant stress and relevant markers. Much attention has focused on identifying specific markers of oxidative stress and inflammation that could be measured in easily accessible tissues and fluids (lymphocytes, plasma, serum). The purpose of this review is to discuss markers of oxidant stress used in the field as biomarkers of aging and age-related diseases, highlighting differences observed by race when data is available. We highlight DNA, RNA, protein, and lipid oxidation as measures of oxidative stress, as well as other well-characterized markers of oxidative damage and inflammation and discuss their strengths and limitations. We present the current state of the literature reporting use of these markers in studies of human cohorts in relation to age and age-related disease and also with a special emphasis on differences observed by race when relevant.

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1).

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.

microRNA expression patterns reveal differential expression of target genes with age.

Recent evidence supports a role for microRNAs (miRNAs) in regulating the life span of model organisms. However, little is known about how these small RNAs contribute to human aging. Here, we profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.