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Purpose: Lung adenocarcinoma (LUAD) is a clinically heterogeneous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated with immunotherapies, which cannot be explained by mutational profiles. DNA methylation–based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs. Experimental Design: We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups. Results: Deconvolution of DNA methylation data identified immunologically hot and cold subsets of LUADs. In addition, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified six molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics. Conclusions: Our results demonstrate that DNA methylation signatures can stratify LUAD into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)—key downstream effectors of LKB1— induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer.SIGNIFICANCELoss-of-function mutations in theLKB1tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

Supplementary Data from DNA Methylation Profiling Identifies Subgroups of Lung Adenocarcinoma with Distinct Immune Cell Composition, DNA Methylation Age, and Clinical Outcome

Purpose:Lung adenocarcinoma (LUAD) is a clinically heterogeneous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated with immunotherapies, which cannot be explained by mutational profiles. DNA methylation–based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs.Experimental Design:We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups.Results:Deconvolution of DNA methylation data identified immunologically hot and cold subsets of LUADs. In addition, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified six molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics.Conclusions:Our results demonstrate that DNA methylation signatures can stratify LUAD into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.

Supplementary Figure from DNA Methylation Profiling Identifies Subgroups of Lung Adenocarcinoma with Distinct Immune Cell Composition, DNA Methylation Age, and Clinical Outcome

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP(8)). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP(8) synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP(8)) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP(8) levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP(8); PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP(8) to the XPR1 N-terminus (K(d) = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

Objective: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE −/− and TSP-1 −/− /ApoE −/− double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE −/− mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE −/− mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE −/− ; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE −/− on western diet, independent of plasma lipid alterations. Conclusions: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

OBJECTIVE –: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on thrombospondin-1 (TSP-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. APPROACH AND RESULTS –: 17-wks-old ApoE(−/−) and TSP-1(−/−)/ApoE(−/−) double knockout (dKO) mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5µg/g bwt, IP) once daily for three weeks. Using aortic root morphometry and en-face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area and collagen accumulation in aortic roots of ApoE(−/−) mice, shown via Oil red O, H & E and Masson’s trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE(−/−) mice. Moreover, these effects were concomitant to changes in VLDL-triglyceride and HDL-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB accompanied with augmented SM-MHC expression in aortic vessels of leptin-treated dKO vs. leptin-treated ApoE(−/−); also confirmed in aortic SMCs from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE(−/−) on western diet, independent of plasma lipid alterations. CONCLUSIONS –: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced VSMC phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP(7) (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP(7) inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP(7) levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP(7) synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP(7) analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP(7) levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

Atherosclerosis is the leading cause of increased morbidity and mortality in metabolic syndrome (MetS), a constellation of risk factors that include hyperglycemia and visceral obesity. Individuals with MetS have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a key characteristic of VSMC phenotypic transition. We previously reported that high glucose and high leptin, mimicking hyperglycemia and obesity, independently upregulate a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), expression in VSMCs. The goal of the present study was to investigate the role of TSP-1 in development of atherosclerosis in MetS. We generated a mouse model of combined MetS and atherosclerosis ( KKAy +/- /ApoE -/- ) by crossing obese hyperglycemic agouti yellow KKAy +/- mice with atherosclerotic ApoE -/- mice. Upon weaning, yellow KKAy +/- /ApoE -/- mice and age-matched black KKAy -/- /ApoE -/- littermates were maintained on regular chow diet from 4-18 wks of age. Mice were euthanized after an overnight fasting; blood, aortae and heart were collected for lipid profiling, immunoblotting and atherosclerotic lesion studies. Yellow KKAy +/- /ApoE -/- mice showed significant increase in body weight, blood glucose and plasma lipid levels vs. black KKAy -/- /ApoE -/- littermates. Aortic root morphometry demonstrated increased lesion burden (Oil red O) and neointimal thickening (H & E) in MetS KKAy +/- /ApoE -/- vs. non-MetS KKAy -/- /ApoE -/- mice. Immunohistochemistry further revealed co-localization of α-SMA (SMC marker) and PCNA (proliferation marker) expression in aortic roots of KKAy +/- /ApoE -/- vs. KKAy -/- /ApoE -/- mice. Notably, lesion formation was associated with an increase in TSP-1 expression in aortic vessels. Consistently, TSP-1 and PCNA expression were elevated in aortic SMC (aSMC) primary cultures derived from obese diabetic KKAy +/- mice vs. lean non-diabetic KKAy -/- littermates. Finally, incubation of aSMC with a TSP-1 blocking peptide reduced PCNA and vimentin (SMC synthetic marker) expression coupled with attenuated SMC proliferation in obese diabetic KKAy +/- mice. Together, these results suggest a putative role of TSP-1 in accelerated atherosclerosis and VSMC phenotypic transition in MetS.

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE−/−) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE−/− mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1−/−/ApoE−/− double knockout mice were compared with age-matched hyperglycemic ApoE−/− littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE−/− mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

Atherosclerosis, a major macrovascular complication associated with diabetes, poses a tremendous burden on national health care expenditure. Despite extensive efforts, cost-effective remedies are unknown. Therapies for atherosclerosis are challenged by a lack of targeted drug delivery approaches. Toward this goal, we turn to a biology-derived drug delivery system utilizing nanoparticles formed by the plant virus, Cowpea mosaic virus (CPMV). The aim herein is to investigate the anti-atherogenic potential of the beneficial mineral nutrient, trivalent chromium, loaded CPMV nanoparticles in human aortic smooth muscle cells (HASMC) under hyperglycemic conditions. A non-covalent loading protocol is established yielding CrCl3-loaded CPMV (CPMV-Cr) carrying 2000 drug molecules per particle. Using immunofluorescence microscopy, we show that CPMV-Cr is readily taken up by HASMC in vitro. In glucose (25 mM)-stimulated cells, 100 nM CPMV-Cr inhibits HASMC proliferation concomitant to attenuated proliferating cell nuclear antigen (PCNA, proliferation marker) expression. This is accompanied by attenuation in high glucose-induced phospho-p38 and pAkt expression. Moreover, CPMV-Cr inhibits the expression of pro-inflammatory cytokines, transforming growth factor-β (TGF-β) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), in glucose-stimulated HASMCs. Finally glucose-stimulated lipid uptake is remarkably abrogated by CPMV-Cr, revealed by Oil Red O staining. Together, these data provide key cellular evidence for an atheroprotective effect of CPMV-Cr in vascular smooth muscle cells (VSMC) under hyperglycemic conditions that may promote novel therapeutic ventures for diabetic atherosclerosis.

We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (−1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (−1019 to −1016) and CREB (−1198 to −1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells.

Hyperleptinemia is an important pathogenic contributor of atherosclerosis; however, underlying mechanisms are poorly understood. We reported earlier that leptin upregulates a potent proatherogenic ...

Trivalent chromium (Cr3+) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr3+ supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr3+ in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr3+ (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr3+ occurs at the level of transcription. The inhibitory effects of Cr3+ on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr3+ is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine ( O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr3+ attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr3+ that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr3+ to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications.

Hyperleptinemia, characteristic of diabetes and a hallmark feature of obesity, contributes to the increased risk of atherosclerotic complications. We recently reported that leptin has a direct regulatory effect on the expression of a potent proatherogenic and antiangiogenic matricellular protein thrombospondin-1 (TSP-1) in vitro and in vivo . While leptin and TSP-1 are independently linked to atherosclerosis, a potential cross-talk in vivo remains unknown. The present study is the first evidence that TSP-1 plays a direct role in leptin-induced atherosclerosis. Briefly ApoE -/- and TSP-1 -/- /ApoE -/- double knockout (dKO) mice at 17 wks of age, on regular chow diet, were injected i.p. with murine recombinant leptin (5μg/g body weight) or PBS (vehicle control) once daily for 3 wks and mice were sacrificed at 20 wks of age. Using en face atherosclerotic lesion assay shown by Oil red O staining, we demonstrated that while leptin in vivo significantly increased (2.5-fold) lesion area in the aortic walls of ApoE -/- mice, TSP-1 -/- /ApoE -/- dKO mice developed relatively fewer lesions in response to exogenous leptin. Using high-frequency ultrasound imaging we found that in ApoE -/- mice leptin significantly decreased diameter of transaortic arch, ascending and descending aorta (p < 0.01), while TSP-1 -/- /ApoE -/- dKO mice were completely protected against this leptin-induced reduction in aortic vessel diameter. Ultrasound imaging of common carotid artery further revealed that leptin-treated TSP-1 -/- /ApoE -/- dKO mice were protected against development of vascular lesions while leptin-treated ApoE -/- mice showed more pronounced lesions and a significant reduction in carotid artery internal diameter (p ≤ 0.02). Finally, while leptin in vivo significantly increased plasma cholesterol (~40%) and triglyceride (2.5 fold) levels in ApoE -/- mice, this leptin-induced increase in plasma lipid levels was attenuated in TSP-1 -/- /ApoE -/- dKO mice. Together, our results suggest that knockout of TSP-1 protects ApoE -/- mice against leptin-induced atherosclerosis in vivo , underscoring a novel role of TSP-1 in hyperleptinemia-induced macrovascular complications.

Vascular disease accounts for increased morbidity and mortality in diabetes. Given the burden of health care expenditure in diabetics, relatively cost-effective strategies represent an attractive therapeutic approach. Trivalent chromium (Cr) is a mineral nutrient reported to have a beneficial role in glycemic and cardiovascular health. While previous studies suggest that Cr reduces vascular inflammation in diabetes, the precise mechanisms of Cr action in the large blood vessels in diabetes remain unknown. We showed earlier that Cr picolinate (CrP, a bioavailable form of Cr) in vitro downregulates a potent proatherogenic matricellular protein thrombospondin-1 (TSP-1) in glucose-stimulated vascular smooth muscle cells. The present study provides the first evidence of an atheroprotective mechanism of Cr in vivo in a mouse model of diabetic atherosclerosis. Briefly, hyperglycemia was induced in male ApoE -/- mice (6wks age) with 50mg/Kg/day streptozotocin (STZ), i.p. for 5 consecutive days. Hyperglycemic mice (blood glucose>250mg/dl) on regular chow diet were treated with or without CrP (8μg/Kg) in drinking water starting at 8 wks of age and all mice were sacrificed at 18 wks of age. Using en face atherosclerotic lesion assay shown by Oil red O staining, we demonstrated that CrP in vivo significantly prevents development of lesion in aortic vessel walls of hyperglycemic STZ-ApoE -/- mice compared to untreated STZ-ApoE -/- mice. Consistently, high-frequency ultrasound imaging of common carotid artery revealed that CrP prevents development of vascular lesions in STZ-ApoE -/- mice while untreated mice showed more pronounced lesions with a significant reduction (20%) in carotid artery diameter. Importantly, atheroprotective effects of CrP were accompanied by a significant decrease (>35%) in TSP-1 and PCNA (proliferation marker) expression in aortic vessels of STZ-ApoE -/- mice, shown by immunoblotting. Also, en face studies showed that knockout of TSP-1 protects STZ-induced hyperglycemic ApoE -/- mice against atherosclerosis. Together, our results suggest that CrP in vivo prevents diabetic atherosclerosis via downregulation of TSP-1 in the large blood vessel wall, underscoring TSP-1 as a novel therapeutic target in diabetic atherosclerosis.