Faculty Bibliography

RAGE and its intracellular effector molecule, the actin polymerase DIAPH1, mediate inflammation and the complications of diabetes. Using NMR spectroscopy and mass spectrometry, we built a structural model of the RAGE-DIAPH1 complex, revealing how binding of the cytoplasmic tail of RAGE (ctRAGE) to DIAPH1 stimulates its actin polymerization activity, which is inhibited by a small molecule antagonist of RAGE-DIAPH1 interaction, RAGE406R. The solution structure of the RAGE406R - ctRAGE suggests that RAGE406R prevents the formation of the RAGE-DIAPH1. FRET, actin polymerization assays, smooth muscle cell migration, and THP1 cell inflammation experiments, together with the in vivo interrogation of the effects of RAGE406R in mouse models of inflammation and diabetic wound healing, support this mode of RAGE-DIAPH1 antagonism. Finally, the treatment of macrophages differentiated from peripheral blood-derived mononuclear cells from humans with type 1 diabetes with RAGE406R reduces the mRNA expression of the chemokine CCL2, diminishing the expression of a key node in the inflammatory response.

Ischemic heart disease is the leading cause of heart failure with reduced ejection fraction in the developed world. An evolution of background medical therapy over the past decade has spurred improvement in symptoms and a reduction in morbidity and mortality with ischemic cardiomyopathy. However, there is still ongoing debate about the role and impact of revascularization. Much of the societal guidance regarding revascularization with coronary artery bypass grafting in ischemic cardiomyopathy comes from the STICH trial (Surgical Treatment for Ischemic Heart Failure) which predates improvements in medical therapy. More recently, the REVIVED-BCIS2 trial (Revascularization for Ischemic Ventricular Dysfunction-British Cardiovascular Intervention Society) failed to show a benefit of percutaneous coronary intervention on heart failure hospitalization and mortality in ischemic cardiomyopathy over contemporary medical therapy alone. Yet, there are outstanding questions regarding the role and modality of revascularization required to improve outcomes. We review current data and future directions in the management of ischemic cardiomyopathy and the potential role of revascularization.

OBJECTIVE:This study examined racial and ethnic differences in percent total weight loss (%TWL) and glycemic improvement following sleeve gastrectomy (SG) and explored the role of socioeconomic and psychosocial factors in postsurgical outcomes. METHODS:This longitudinal study included patients who underwent SG between 2017 and 2020, with follow-up visits over 24 months. RESULTS:Non-Hispanic Black (NHB) participants had lower %TWL at 3, 12, and 24 months compared with Hispanic (H) and non-Hispanic White (NHW) participants. Fat mass index was initially lower in NHB, with smaller reductions over time and significant group differences persisting at 24 months. NHB participants had higher baseline fat-free mass index values; by 24 months, fat-free mass index values were lower in H participants. Hemoglobin A1c decreased across all groups but remained consistently higher in NHB and H compared with NHW at 24 months. NHB participants reported higher perceived discrimination, sleep disturbance, and perceived stress than H and NHW participants at all time points. Employment status predicted %TWL at 12 months. There was a significant interaction between race and ethnicity and employment status observed at 12 and 24 months, suggesting that employment-related disparities could impact surgical outcomes. CONCLUSIONS:NHB participants experienced less favorable outcomes following SG, emphasizing the need for tailored interventions addressing socioeconomic and psychosocial disparities.

DIAPH1 is a member of the family of Diaphanous Related Formins (DRFs) implicated in cell migration and cytokinesis. DRFs are maintained in an autoinhibited state by the intramolecular association between diaphanous inhibitory (DID) and diaphanous autoregulatory (DAD) domains. Actin polymerization requires the binding of activated RhoA to the GTPase binding domain (GBD) of DIAPH1 and the dissociation of DAD. In the presence of excess RhoA, actin polymerization is only partially activated. Using monomeric domain constructs of DIAPH1, the sequential binding affinities of RhoA and DAD to GBD-DID were characterized. Binding of RhoA and DAD were negatively cooperative requiring a 100-fold greater concentration of DAD to achieve saturation when RhoA binding site was occupied. The unimolecular architecture of full length DIAPH1 establishes an effective concentration of DAD in the micromolar range, which is 100-fold larger than the intrinsic affinity of DAD for DID. The effective concentration is large enough to maintain DIAPH1 autoinhibition, yet small enough to permit partial activation of DIAPH1 after RhoA binding. By exploiting negative cooperativity, DIAPH1 maintains a reserve of inactivated molecules enabling gradual responses to cellular processes that require prolonged and sustained regulation. The proposed mechanism is extended to other DIAPH1 activating ligands and broadly applicable to all DRFs.

Diabetes is a major risk factor for cardiovascular diseases. Patients with diabetes are at greater risk for morbidity and mortality post myocardial infarction. As the epidemic of diabetes continues at an alarming pace, identification of specific therapeutic interventions to protect diabetic patients from the devastating consequences of myocardial infarction is an urgent need. Advanced glycation end products (AGEs), the products of nonenzymatic glycation and oxidation of proteins and lipids, accumulate in the diabetic circulation and heart. The interaction of AGEs with its key receptor, receptor for AGE or RAGE, contributes to cardiac injury and dysfunction. The discovery that intracellular domain of RAGE binds to the formin, DIAPH1, and that DIAPH1 is essential for RAGE ligand-mediated signal transduction, unveiled the specific cellular means by which RAGE functions and highlights a new target for therapeutic interruption of pathological RAGE signaling during myocardial infarction. This review delves into intrinsic mechanisms by which AGE-RAGE axis via RAGE-DIAPH1 driven DIAPH1-Mitofusin2 (MFN2) interaction modulates pathogenic inter-organelle communications and opens opportunities for intensive studies to uncover the comprehensive mechanisms that drive injury-provoking actions from the intracellular space. This review illustrates the potential therapeutic cardioprotective benefits of antagonism of RAGE-DIAPH1interactions in the diabetic heart.

BACKGROUND & AIMS/UNASSIGNED:Metabolic syndrome-associated steatotic liver disease (MASLD) and metabolic syndrome-associated steatohepatitis (MASH) have global prevalence rates exceeding 25% and 3-6%, respectively. The introduction of high-fructose corn syrup to the diet in the 1970s has been linked to metabolic and hepatic disturbances. Despite these associations, the potential for sex-dependent responses resulting from fructose-containing diets on MASLD/MASH has not been addressed. METHODS/UNASSIGNED:standard chow for 16 weeks (n = 40 mice). At sacrifice, plasma and liver were retrieved, the latter for single-nucleus RNA sequencing. Publicly available data sets of human male and female MASH liver were probed. RESULTS/UNASSIGNED:0.0001). Single-nucleus RNA sequencing revealed distinct sex-specific transcriptional profiles in hepatocytes and stellate cells responding to the FPC-NASH diet compared to the standard chow. In female mice, compared to males, pathways associated with lipid and metabolic processes in hepatocytes and cell-cell communication and adhesion in stellate cells were enriched. Metabolic flux analyses demonstrated reduced bile acid metabolism in female mice and human hepatocytes in FPC-NASH and MASH conditions, respectively, compared to their male counterparts. CONCLUSIONS/UNASSIGNED:Molecular profiling of hepatocytes and stellate cells in FPC-NASH diet-fed mice revealed significant sex differences mirrored in human MASH. The identification of intrinsic, within-sex, diet-dependent disparities underscores the critical need to include both male and female individuals in MAFLD/MASH studies and clinical trials. IMPACT AND IMPLICATIONS/UNASSIGNED:male patients with MASH. These results highlight potential mechanistic explanations and therapeutic targets for addressing sex differences and underscore the need to study both sexes in animal models and human MASH.

The advanced glycation end products (AGEs) Webinar was co-hosted by Diabetes Technology Society and Kitalys Institute on August 8, 2024, with the goal of reviewing progress made in the measurement and use of AGEs in clinical practice. Meeting topics included (1) AGEs as predictors of diabetic nephropathy (DKD), (2) hemoglobin glycation index (HGI) and the glycation gap (GG), (3) formation and structure of AGEs, (4) AGEs as a risk factor of cardiovascular disease (CVD), and (5) approaches to limit or prevent AGE formation.

Diabetes affects metabolism and metabolite concentrations in multiple organs. Previous preclinical studies have shown that receptor for advanced glycation end products (RAGE, gene symbol Ager) and its cytoplasmic domain binding partner, Diaphanous-1 (DIAPH1), are key mediators of diabetic micro- and macro-vascular complications. In this study, we used ¹H-Magnetic Resonance Spectroscopy (MRS) and chemical shift encoded (CSE) Magnetic Resonance Imaging (MRI) to investigate the metabolite and water-fat fraction in the heart and hind limb muscle in a murine model of type 1 diabetes (T1D) and to determine if the metabolite changes in the heart and hind limb are influenced by (a) deletion of Ager or Diaph1 and (b) pharmacological blockade of RAGE-DIAPH1 interaction in mice. Nine cohorts of male mice, with six mice per cohort, were used: wild type non-diabetic control mice (WT-NDM), WT-diabetic (WT-DM) mice, Ager knockout non-diabetic (RKO-NDM) and diabetic mice (RKO-DM), Diaph1 knockout non-diabetic (DKO-NDM), and diabetic mice (DKO-DM), WT-NDM mice treated with vehicle, WT-DM mice treated with vehicle, and WT-DM mice treated with RAGE229 (antagonist of RAGE-DIAPH1 interaction). A Point Resolved Spectroscopy (PRESS) sequence for ¹H-MRS, and multi-echo gradient recalled echo (GRE) for CSE were employed. Triglycerides, and free fatty acids in the heart and hind limb obtained from MRS and MRI were compared to those measured using biochemical assays. Two-sided t-test, non-parametric Kruskal-Wallis Test, and one-way ANOVA were employed for statistical analysis. We report that the results were well-correlated with significant differences using MRI and biochemical assays between WT-NDM and WT-DM, as well as within the non-diabetic groups, and within the diabetic groups. Deletion of Ager or Diaph1, or treatment with RAGE229 attenuated diabetes-associated increases in triglycerides in the heart and hind limb in mice. These results suggest that the employment of ¹H-MRS/MRI is a feasible non-invasive modality to monitor metabolic dysfunction in T1D and the metabolic consequences of interventions that block RAGE and DIAPH1.

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: : Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. Compared with that in control mice, the protein in these mice clears from the serum faster and accumulates significantly more in fibrotic livers. This work advances the development of targeted protein probes for in vivo fibrosis imaging.