Islet amyloid polypeptide toxicity and membrane interactions.

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of ß-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Messenger RNA in lipid nanoparticles rescues HEK 293 cells from lipid-induced mitochondrial dysfunction as studied by real time pulse chase NMR, RTPC-NMR, spectroscopy.

Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured cells.

Aldose Reductase: An Emerging Target for Development of Interventions for Diabetic Cardiovascular Complications.

Diabetes is a leading cause of cardiovascular morbidity and mortality. Despite numerous treatments for cardiovascular disease (CVD), for patients with diabetes, these therapies provide less benefit for protection from CVD. These considerations spur the concept that diabetes-specific, disease-modifying therapies are essential to identify especially as the diabetes epidemic continues to expand. In this context, high levels of blood glucose stimulate the flux via aldose reductase (AR) pathway leading to metabolic and signaling changes in cells of the cardiovascular system. In animal models flux via AR in hearts is increased by diabetes and ischemia and its inhibition protects diabetic and non-diabetic hearts from ischemia-reperfusion injury. In mouse models of diabetic atherosclerosis, human AR expression accelerates progression and impairs regression of atherosclerotic plaques. Genetic studies have revealed that single nucleotide polymorphisms (SNPs) of the ALD2 (human AR gene) is associated with diabetic complications, including cardiorenal complications. This Review presents current knowledge regarding the roles for AR in the causes and consequences of diabetic cardiovascular disease and the status of AR inhibitors in clinical trials. Studies from both human subjects and animal models are presented to highlight the breadth of evidence linking AR to the cardiovascular consequences of diabetes.

Porphyromonas gingivalis infection and prothrombotic effects in human aortic smooth muscle cells.

INTRODUCTION: Accumulating evidence has demonstrated an association between periodontal infectious agents, such as Porphyromonas gingivalis, and vascular disease. Tissue factor (TF) and its specific tissue factor pathway inhibitor (TFPI) are produced by vascular cells and are important regulators of the coagulation cascade. MATERIALS AND METHODS: To assess the role of P. gingivalis in atherothrombosis, we infected primary human aortic smooth muscle cells (HASMC) with either P. gingivalis 381, its non-invasive mutant DPG3, or heat-killed P. gingivalis 381. Levels and activity of TF and TFPI were measured 8 and 24 hours after infection in cell extracts and cell culture supernatants. RESULTS: P. gingivalis 381 did not affect total TF antigen or TF activity in HASMC, but it significantly suppressed TFPI levels and activity compared to uninfected control cells, and those infected with the non-invasive mutant strain or the heat-killed bacteria. Further, P. gingivalis' LPS (up to a concentration of 5 microg/ml) failed to induce prothrombotic effects in HASMC, suggesting a significant role for the ability of whole viable bacteria to invade this cell type. CONCLUSION: These data demonstrate for the first time that infection with a periodontal pathogen induces a prothrombotic response in HASMC.

Incense Burning is Associated with Human Oral Microbiota Composition.

Incense burning is common worldwide and produces environmental toxicants that may influence health; however, biologic effects have been little studied. In 303 Emirati adults, we tested the hypothesis that incense use is linked to compositional changes in the oral microbiota that can be potentially significant for health. The oral microbiota was assessed by amplification of the bacterial 16S rRNA gene from mouthwash samples. Frequency of incense use was ascertained through a questionnaire and examined in relation to overall oral microbiota composition (PERMANOVA analysis), and to specific taxon abundances, by negative binomial generalized linear models. We found that exposure to incense burning was associated with higher microbial diversity (p < 0.013) and overall microbial compositional changes (PERMANOVA, p = 0.003). Our study also revealed that incense use was associated with significant changes in bacterial abundances (i.e. depletion of the dominant taxon Streptococcus), even in occasional users (once/week or less) implying that incense use impacts the oral microbiota even at low exposure levels. In summary, this first study suggests that incense burning alters the oral microbiota, potentially serving as an early biomarker of incense-related toxicities and related health consequences. Although a common indoor air pollutant, guidelines for control of incense use have yet to be developed.

Infection with a periodontal pathogen increases mononuclear cell adhesion to human aortic endothelial cells.

BACKGROUND: As a link between periodontal infections and an increased risk for vascular disease has been demonstrated, we assessed the ability of the Gram-negative periodontal pathogen Porphyromonas gingivalis to modulate properties of endothelial cells linked to inflammation and proatherogenic pathways. METHODS AND RESULTS: Primary human aortic endothelial cells (HAEC) were infected with either P. gingivalis strain 381 or its non-invasive fimbriae-deficient mutant, DPG3, and incubated with U-937 monocytes, or Jurkat T cells. P. gingivalis-infected HAEC demonstrated significantly increased adhesion of immune cells compared to non-infected cells or those infected with DPG3. Heat-killed bacteria had no effect on mononuclear cell adhesion and P. gingivalis LPS had only a minimal effect. P. gingivalis infection significantly increased HAEC expression of VCAM-1, ICAM-1 and E-selectin, and enhanced production of IL-6, IL-8 and MCP-1. CONCLUSION: These data demonstrate that live invasive P. gingivalis 381 elicits a pro-atherogenic response in HAEC.

Porphyromonas gingivalis infection and cell death in human aortic endothelial cells.

Porphyromonas gingivalis is a periodontal pathogen that promotes a proatherogenic response in endothelial cells. Cell death responses of human aortic endothelial cells to P. gingivalis at various multiplicities of infection (MOI) were investigated by assessment of cell detachment, histone-associated DNA fragmentation, lactate dehydrogenase release and ADP:ATP ratio. Porphyromonas gingivalis at MOI 1:10-1:100 did not have a cytotoxic effect, but induced apoptotic cell death at MOI 1:500 and 1:1000. Monocyte chemoattractant protein-1 production was significantly enhanced by P. gingivalis at MOI 1:100. At higher MOI, at least in vitro, P. gingivalis mediates endothelial apoptosis, thereby potentially amplifying proatherogenic mechanisms in the perturbed vasculature.

Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice.

OBJECTIVE: Because recent epidemiologic evidence suggests that periodontal infections may increase the risk of atherosclerosis and related events in humans, we assessed the impact of oral inoculation with the periodontal pathogen Porphyromonas gingivalis on atherogenesis in hypercholesterolemic apolipoprotein E-null mice. METHODS AND RESULTS: In the absence of alterations in distinct risk factors, P gingivalis infection exacerbated the early stages of atherogenesis in this model. Infected animals displayed evidence of local periodontal infection, as the severity of alveolar bone loss, the hallmark of periodontitis, was increased. Generalized activation of host inflammatory responses was evident in infected mice, as demonstrated by serum IgG response to P gingivalis and elevated levels of interleukin-6. P gingivalis DNA was localized in the aortic tissue from a limited number of infected mice but not in any noninfected controls. Infected mice displayed enhanced vascular activation, as suggested by increased aortic expression of vascular cell adhesion molecule-1 and tissue factor. CONCLUSIONS: Oral infection with P gingivalis accelerates early atherosclerosis. Thus, uncovering the underlying mechanisms is critical for the design of preventive and therapeutic strategies targeting atherosclerotic vascular disease and its sequelae.

Porphyromonas gingivalis induces its uptake by human macrophages and promotes foam cell formation in vitro.

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans, which has been linked to an increased risk for atherosclerosis-related events. In this study, we examined the effect of P. gingivalis infection on human macrophages with respect to foam cell formation, the hallmark of early atherogenesis, and the potential of P. gingivalis to induce its uptake by these cells. Human monocyte-derived macrophages were incubated with low density lipoprotein and infected with P. gingivalis FDC381 or its fimbriae deficient mutant, DPG3. Consistent with a role for fimbriae in this process, strain 381 significantly increased foam cell formation as compared to DPG3. Recovery of viable P. gingivalis in antibiotic protection experiments was significantly higher for strain 381 than for DPG3. By transmission electron microscopy, the wild-type strain was shown to adhere to and enter THP-1 cells. These results suggest that properties of P. gingivalis which render it capable of adhering to/invading other cell types may also be operative in macrophages and play an important role in its atherogenic potential.

Carbon monoxide form of PEGylated hemoglobin protects myocardium against ischemia/reperfusion injury in diabetic and normal mice.

We investigated the pre-clinical utility of carbon monoxide form of PEGylated hemoglobin (PEG-Hb also named SANGUINATE(™)) in myocardial infarction (MI) and in particular the response of diabetic tissues to superimposed ischemia/reperfusion injury. SANGUINATE(™) was evaluated in diabetic and normal mice subjected to 30 min of coronary artery ligation followed by either 48 h or 28 days of reperfusion. Our results demonstrate that SANGUINATE(™) was effective in reducing infarct size when administered either prior to left anterior descending coronary artery (LAD) occlusion or during reperfusion. This finding is an important step in exploring the efficacy of a pharmacoinvasive strategy using SANGUINATE(™) in patients with acute coronary syndromes.

Diabetes and oral disease: implications for health professionals.

"Diabetes and Oral Disease: Implications for Health Professionals" was a one-day conference convened by the Columbia University College of Dental Medicine, the Columbia University College of Physicians and Surgeons, and the New York Academy of Sciences on May 4, 2011 in New York City. The program included an examination of the bidirectional relationship between oral disease and diabetes and the interprofessional working relationships for the care of people who have diabetes. The overall goal of the conference was to promote discussion among the healthcare professions who treat people with diabetes, encourage improved communication and collaboration among them, and, ultimately, improve patient management of the oral and overall effects of diabetes. Attracting over 150 members of the medical and dental professions from eight different countries, the conference included speakers from academia and government and was divided into four sessions. This report summarizes the scientific presentations of the event.

Receptor for advanced glycation endproducts mediates pro-atherogenic responses to periodontal infection in vascular endothelial cells.

OBJECTIVE: A link between periodontal infections and an increased risk for vascular disease has been demonstrated. Porphyromonas gingivalis, a major periodontal pathogen, localizes in human atherosclerotic plaques, accelerates atherosclerosis in animal models and modulates vascular cell function. The receptor for advanced glycation endproducts (RAGE) regulates vascular inflammation and atherogenesis. We hypothesized that RAGE is involved in P. gingivalis's contribution to pro-atherogenic responses in vascular endothelial cells. METHODS AND RESULTS: Murine aortic endothelial cells (MAEC) were isolated from wild-type C57BL/6 or RAGE-/- mice and were infected with P. gingivalis strain 381. P. gingivalis 381 infection significantly enhanced expression of RAGE in wild-type MAEC. Levels of pro-atherogenic advanced glycation endproducts (AGEs) and monocyte chemoattractant protein 1 (MCP-1) were significantly increased in wild-type MAEC following P. gingivalis 381 infection, but were unaffected in MAEC from RAGE-/- mice or in MAEC infected with DPG3, a fimbriae-deficient mutant of P. gingivalis 381. Consistent with a role for oxidative stress and an AGE-dependent activation of RAGE in this setting, both antioxidant treatment and AGE blockade significantly suppressed RAGE gene expression and RAGE and MCP-1 protein levels in P. gingivalis 381-infected human aortic endothelial cells (HAEC). CONCLUSION: The present findings implicate for the first time the AGE-RAGE axis in the amplification of pro-atherogenic responses triggered by P. gingivalis in vascular endothelial cells.