Whole-Genome Sequencing of Finnish Type 1 Diabetic Siblings Discordant for Kidney Disease Reveals DNA Variants associated with Diabetic Nephropathy.

BACKGROUND: Several genetic susceptibility loci associated with diabetic nephropathy have been documented, but no causative variants implying novel pathogenetic mechanisms have been elucidated. METHODS: We carried out whole-genome sequencing of a discovery cohort of Finnish siblings with type 1 diabetes who were discordant for the presence (case) or absence (control) of diabetic nephropathy. Controls had diabetes without complications for 15-37 years. We analyzed and annotated variants at genome, gene, and single-nucleotide variant levels. We then replicated the associated variants, genes, and regions in a replication cohort from the Finnish Diabetic Nephropathy study that included 3531 unrelated Finns with type 1 diabetes. RESULTS: We observed protein-altering variants and an enrichment of variants in regions associated with the presence or absence of diabetic nephropathy. The replication cohort confirmed variants in both regulatory and protein-coding regions. We also observed that diabetic nephropathy-associated variants, when clustered at the gene level, are enriched in a core protein-interaction network representing proteins essential for podocyte function. These genes include protein kinases (protein kinase C isoforms ε and ι) and protein tyrosine kinase 2. CONCLUSIONS: Our comprehensive analysis of a diabetic nephropathy cohort of siblings with type 1 diabetes who were discordant for kidney disease points to variants and genes that are potentially causative or protective for diabetic nephropathy. This includes variants in two isoforms of the protein kinase C family not previously linked to diabetic nephropathy, adding support to previous hypotheses that the protein kinase C family members play a role in diabetic nephropathy and might be attractive therapeutic targets.

P-selectin glycoprotein ligand-1 regulates adhesive properties of the endothelium and leukocyte trafficking into adipose tissue.

RATIONALE: Adhesive interactions between endothelial cells and leukocytes affect leukocyte trafficking in adipose tissue. The role of P-selectin glycoprotein ligand-1 (Psgl-1) in this process is unclear. OBJECTIVE: The goal of this study was to determine the effect of Psgl-1 deficiency on adhesive properties of the endothelium and on leukocyte recruitment into obese adipose depots. METHODS AND RESULTS: A genetic model of obesity was generated to study the effects of Psgl-1 deficiency on leukocyte trafficking. Leukocyte-endothelial interactions were increased in obese leptin receptor mutant mice (Lepr(db/db),Psgl-1(+/+)) but not obese Psgl-1-deficient mice (Lepr(db/db),Psgl-1(-/-)), when compared with lean mice (Lepr(+/+),Psgl-1(+/+)). This effect of Psgl-1 deficiency was due to indirect effects of Psgl-1, because Psgl-1(+/+) adoptively transferred leukocytes did not exhibit enhanced rolling in Lepr (db/db),Psgl-1(-/-) mice. Additionally, circulating levels of P-selectin, E-selectin, monocyte chemoattractant protein-1, and macrophage content of visceral adipose tissue were reduced in Lepr(db/db),Psgl-1(-/-) compared with Lepr(db/db),Psgl-1(+/+) mice. Reduced leukocyte-endothelial interactions and macrophage content of visceral adipose tissue due to Psgl-1 deficiency was also observed in a diet-induced obese mouse model. Psgl-1(-/-) mice were resistant to the endothelial effects of exogenous IL-1beta, suggesting that defective cytokine signaling contributes to the effect of Psgl-1 deficiency on leukocyte-endothelial interactions. Mice deficient in the IL-1 receptor also had reduced levels of circulating P-selectin, similar to those observed in Psgl-1(-/-) mice. CONCLUSIONS: Deficiency of Psgl-1 is associated with reduced IL-1 receptor-mediated adhesive properties of the endothelium and is protective against visceral fat inflammation in obese mice.

Monocyte chemoattractant protein-1 deficiency protects against visceral fat-induced atherosclerosis.

OBJECTIVE: To determine the role of monocyte chemoattractant protein-1 (Mcp-1) on the progression of visceral fat-induced atherosclerosis. METHODS AND RESULTS: Visceral fat inflammation was induced by transplantation of perigonadal fat. To determine whether recipient Mcp-1 status affected atherosclerosis induced by inflammatory fat, apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-) and Mcp-1-deficient (Mcp-1(-/-)) mice underwent visceral fat transplantation. Intravital microscopy was used to study leukocyte-endothelial interactions. To study the primary tissue source of circulating Mcp-1, both fat and bone marrow transplantation experiments were used. Transplantation of visceral fat increased atherosclerosis in ApoE(-/-) mice but had no effect on atherosclerosis in ApoE(-/-),Mcp-1(-/-) mice. Intravital microscopy revealed increased leukocyte attachment to the endothelium in ApoE(-/-) mice compared with ApoE(-/-),Mcp-1(-/-) mice after receiving visceral fat transplants. Transplantation of visceral fat increased plasma Mcp-1, although donor adipocytes were not the source of circulating Mcp-1 because no Mcp-1 was detected in plasma from ApoE(-/-),Mcp-1(-/-) mice transplanted with Wt fat, indicating that recipient Mcp-1-producing cells were affecting the atherogenic response to the fat transplantation. Consistently, transplantation of Mcp-1(-/-) fat to ApoE(-/-) mice did not lead to atheroprotection in recipient mice. Bone marrow transplantation between Wt and Mcp-1(-/-) mice indicated that the primary tissue source of circulating Mcp-1 was the endothelium. CONCLUSIONS: Recipient Mcp-1 deficiency protects against atherosclerosis induced by transplanted visceral adipose tissue.

Visceral adipose tissue inflammation accelerates atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Fat inflammation may play an important role in comorbidities associated with obesity such as atherosclerosis. METHODS AND RESULTS: To first establish feasibility of fat transplantation, epididymal fat pads were harvested from wild-type C57BL/6J mice and transplanted into leptin-deficient (Lep(ob/ob)) mice. Fat transplantation produced physiological leptin levels and prevented obesity and infertility in Lep(ob/ob) mice. However, the transplanted fat depots were associated with chronically increased macrophage infiltration with characteristics identical to those observed in fat harvested from obese animals. The inflammation in transplanted adipose depots was regulated by the same factors that have been implicated in endogenous fat inflammation such as monocyte chemoattractant protein-1. To determine whether this inflamed adipose depot could affect vascular disease in mice, epididymal fat depots were transplanted into atherosclerosis-prone apolipoprotein E-deficient ApoE(-/-) mice. Plasma from ApoE(-/-) mice receiving fat transplants contained increased leptin, resistin, and monocyte chemoattractant protein-1 compared with plasma from sham-operated ApoE(-/-) mice. Furthermore, mice transplanted with visceral fat developed significantly more atherosclerosis compared with sham-operated animals, whereas transplants with subcutaneous fat did not affect atherosclerosis despite a similar degree of fat inflammation. Treatment of transplanted ApoE(-/-) mice with pioglitazone decreased macrophage content of the transplanted visceral fat pad and reduced plasma monocyte chemoattractant protein-1. Importantly, pioglitazone also reduced atherosclerosis triggered by inflammatory visceral fat but had no protective effect on atherosclerosis in the absence of the visceral fat transplantation. CONCLUSIONS: Our results indicate that visceral adipose-related inflammation accelerates atherosclerosis in mice. Drugs such as thiazolidinediones might be a useful strategy to specifically attenuate the vascular disease induced by visceral inflammatory fat.

Visceral adipose tissue and atherosclerosis.

Obesity is a risk factor for complications of atherosclerotic vascular disease such as myocardial infarction and stroke. Recent studies have demonstrated that the vascular risk associated with obesity is correlated particularly with visceral adiposity. These clinical observations indicate that various adipose tissue depots may have differential effects on vascular risk. Cellular constituents of adipose tissue secrete cytokines and chemokines that may affect vascular disease. Visceral fat has been demonstrated to express more inflammatory cytokines than subcutaneous fat in obese states. The adipose tissue secretory profile may reflect the influx of macrophages that has been shown to occur with expansion of fat stores. This macrophage infiltration may lead to a chronic low grade, systemic, inflammatory state. Since circulating markers of inflammation are associated with cardiovascular events, the inflammation triggered by adipose tissue may contribute to increased vascular disease. While the vasculopathic effects of visceral obesity may be best treated by weight loss, long term weight loss is difficult to achieve, even with currently available pharmacotherapies. Therapies that target macrophage accumulation in fat or the adipocyte expression profile may be potentially beneficial in reducing the vascular risk associated with obesity. Further characterization of the factors responsible for promoting atherosclerosis in the setting of visceral obesity may lead to new targets for the prevention of atherosclerosis.

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors.

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology.

Culturing functional pancreatic islets on α5-laminins and curative transplantation to diabetic mice.

The efficacy of islet transplantation for diabetes treatment suffers from lack of cadaver-derived islets, islet necrosis and long transfer times prior to transplantation. Here, we developed a method for culturing mouse and human islets in vitro on α5-laminins, which are natural components of islet basement membranes. Adhering islets spread to form layers of 1-3 cells in thickness and remained normoxic and functional for at least 7 days in culture. In contrast, spherical islets kept in suspension developed hypoxia and central necrosis within 16 h. Transplantation of 110-150 mouse islets cultured on α5-laminin-coated polydimethylsiloxane membranes for 3-7 days normalized blood glucose already within 3 days in mice with streptozotocin-induced diabetes. RNA-sequencing of isolated and cultured mouse islets provided further evidence for the adhesion and spreading achieved with α5-laminin. Our results suggest that use of such in vitro expanded islets may significantly enhance the efficacy of islet transplantation treatment for diabetes.

Membrane-Tethered Metalloproteinase Expressed by Vascular Smooth Muscle Cells Limits the Progression of Proliferative Atherosclerotic Lesions.

BACKGROUND: The MMP (matrix metalloproteinase) family plays diverse and critical roles in directing vascular wall remodeling in atherosclerosis. Unlike secreted-type MMPs, a member of the membrane-type MMP family, MT1-MMP (membrane-type 1 MMP; MMP14), mediates pericellular extracellular matrix degradation that is indispensable for maintaining physiological extracellular matrix homeostasis. However, given the premature mortality exhibited by MT1-MMP-null mice, the potential role of the proteinase in atherogenesis remains elusive. We sought to determine the effects of both MT1-MMP heterozygosity and tissue-specific gene targeting on atherogenesis in APOE (apolipoprotein E)-null mice. METHODS AND RESULTS: MT1-MMP heterozygosity in the APOE-null background (Mmp14+/-Apoe-/- ) significantly promoted atherogenesis relative to Mmp14+/+Apoe-/- mice. Furthermore, the tissue-specific deletion of MT1-MMP from vascular smooth muscle cells (VSMCs) in SM22α-Cre(+)Mmp14F/FApoe-/- (VSMC-knockout) mice likewise increased the severity of atherosclerotic lesions. Although VSMC-knockout mice also developed progressive atherosclerotic aneurysms in their iliac arteries, macrophage- and adipose-specific MT1-MMP-knockout mice did not display this sensitized phenotype. In VSMC-knockout mice, atherosclerotic lesions were populated by hyperproliferating VSMCs (smooth muscle actin- and Ki67-double-positive cells) that were characterized by a proinflammatory gene expression profile. Finally, MT1-MMP-null VSMCs cultured in a 3-dimensional spheroid model system designed to mimic in vivo-like cell-cell and cell-extracellular matrix interactions, likewise displayed markedly increased proliferative potential. CONCLUSIONS: MT1-MMP expressed by VSMCs plays a key role in limiting the progression of atherosclerosis in APOE-null mice by regulating proliferative responses and inhibiting the deterioration of VSMC function in atherogenic vascular walls.

CerS2 haploinsufficiency inhibits ß-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance.

Inhibition of ceramide synthesis prevents diabetes, steatosis, and cardiovascular disease in rodents. Six different ceramide synthases (CerS) that differ in tissue distribution and substrate specificity account for the diversity in acyl-chain composition of distinct ceramide species. Haploinsufficiency for ceramide synthase 2 (CerS2), the dominant isoform in the liver that preferentially makes very-long-chain (C22/C24/C24:1) ceramides, led to compensatory increases in long-chain C16-ceramides and conferred susceptibility to diet-induced steatohepatitis and insulin resistance. Mechanistic studies revealed that these metabolic effects were likely due to impaired ß-oxidation resulting from inactivation of electron transport chain components. Inhibiting global ceramide synthesis negated the effects of CerS2 haploinsufficiency in vivo, and increasing C16-ceramides by overexpressing CerS6 recapitulated the phenotype in isolated, primary hepatocytes. Collectively, these studies reveal that altering sphingolipid acylation patterns impacts hepatic steatosis and insulin sensitivity and identify CerS6 as a possible therapeutic target for treating metabolic diseases associated with obesity.

P-selectin glycoprotein ligand-1 deficiency leads to cytokine resistance and protection against atherosclerosis in apolipoprotein E deficient mice.

Adhesive interactions between endothelial cells and leukocytes contribute to atherosclerotic plaque growth. However, mechanism(s) responsible for endothelial priming and deactivation in inflammatory diseases such as atherosclerosis are not clear. Apolipoprotein E deficient mice were generated with deficiency of P-selectin glycoprotein ligand-1 (Psgl-1(-/-), ApoE(-/-)). On both standard chow and Western diet, Psgl-1(-/-), ApoE(-/-) mice were protected against atherosclerosis compared to Psgl-1(+/+), ApoE(-/-) controls. Psgl-1(-/-), ApoE(-/-) mice also showed reduced leukocyte rolling and firm attachment on endothelial cells, however, adoptively transferred Psgl-1(+/+), ApoE(-/-) leukocytes into Psgl-1(-/-), ApoE(-/-) hosts displayed similar reduced rolling as Psgl-1(-/-), ApoE(-/-) leukocytes. Hematopoietic deficiency of Psgl-1 conferred resistance to the effects of interleukin-1ß (IL-1ß) on leukocyte rolling along with reduced circulating levels of sP-sel and sE-sel. Antibody blockade of Psgl-1 also reduced endothelial activation in response to IL-1ß, eliminated leukocyte rolling, and was protective against atherosclerosis in ApoE(-/-) mice. Monocyte depletion with clodronate restored the endothelial response to IL-1ß in Psgl-1(-/-) mice. This study suggests that Psgl-1 deficiency leads to reduced atherosclerosis and adhesive interactions between endothelial cells and leukocytes by indirectly regulating endothelial responses to cytokine stimulation.

Leptin receptor-induced STAT3-independent signaling pathways are protective against atherosclerosis in a murine model of obesity and hyperlipidemia.

AIMS: Leptin is an adipocyte-derived hormone that has been shown to exert both beneficial metabolic effects and potentially adverse vascular effects in preclinical studies. The primary aim of this study was to determine the effects of leptin receptor signaling pathways on atherosclerosis in the setting of obesity and hyperlipidemia. METHODS AND RESULTS: Mice were generated with deficiency of apolipoprotein E (ApoE(-/-)) and either wild-type leptin receptor expression (Lepr(+/+), ApoE(-/-)), mutant leptin receptor expression defective in all leptin receptor signaling pathways (Lepr(db/db), ApoE(-/-)), or mutant leptin receptor expression with selective deficiency of leptin receptor-STAT3 signaling (Lepr(s/s), ApoE(-/-)). At 27 weeks of age (including 7 weeks on a Western diet), Lepr(db/db), ApoE(-/-) developed severe obesity, hypercholesterolemia, and increased atherosclerosis compared to Lepr(+/+), ApoE(-/-) mice. Despite similar obesity and hyperlipidemia to Lepr(db/db), ApoE(-/-) mice, Lepr(s/s), ApoE(-/-) developed less atherosclerosis than Lepr(db/db), ApoE(-/-) mice. Adipose tissue macrophage content, monocyte chemoattractant protein-1 and fatty-acid-binding protein 4 levels were also reduced in Lepr(s/s), ApoE(-/-) mice compared to Lepr(db/db), ApoE(-/-) mice. CONCLUSIONS: In a mouse model of obesity and hyperlipidemia, leptin receptor-mediated STAT3-independent signaling pathways confer protection against atherosclerosis. These differences occur independently of leptin effects on energy balance.

Atherosclerosis and leukocyte-endothelial adhesive interactions are increased following acute myocardial infarction in apolipoprotein E deficient mice.

OBJECTIVE: To determine the effect of myocardial infarction (MI) on progression of atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice. METHODS AND RESULTS: MI was induced following left anterior descending coronary artery (LAD) ligation in wild-type (WT) (n=9) and ApoE-/- (n=25) mice. Compared to sham-operated animals, MI mice demonstrated increased intravascular leukocyte rolling and firm adhesion by intravital microscopy, reflecting enhanced systemic leukocyte-endothelial interactions. To determine if MI was associated with accelerated atherogenesis, LAD ligation was performed in ApoE-/- mice. Six weeks following surgery, atherosclerosis was quantitated throughout the arterial tree by microdissection and Oil-Red-O staining. There was 1.6-fold greater atherosclerotic burden present in ApoE-/- MI mice compared to sham-operated mice. CONCLUSIONS: Acute MI accelerates atherogenesis in mice. These results may be related to the increased risk of recurrent ischemic coronary events following MI in humans.

Targeting MCP-1 to reduce vascular complications of obesity.

Obesity is a risk factor for complications of atherosclerotic vascular disease such as myocardial infarction. Recent studies and several patents have demonstrated that the cardiovascular risk associated with obesity is correlated particularly with visceral adiposity. Excess visceral adiposity may increase vascular risk due to secretion of cytokines and chemokines by cellular constituents of the adipose tissue. The secretory profile of various adipose depots may be regulated by the influx of macrophages that has been shown to occur with expansion of fat stores. This macrophage infiltration may lead to a chronic low grade, systemic, inflammatory state. Since circulating markers of inflammation are associated with cardiovascular events, the inflammation triggered by visceral fat may contribute to an increased risk for vascular complications. While the vasculopathic effects of central obesity may be best treated by weight loss, long term weight loss is difficult to achieve, even with currently available pharmacotherapies. Therapies that target macrophage accumulation in fat or secretory products of adipose tissue may be potentially beneficial in reducing the vascular risk associated with obesity. A potential therapeutic target is monocyte chemoattractant 1 (MCP-1), which is a potent chemokine that is elevated in obesity. Since MCP-1 promotes atherosclerosis, inhibition of MCP-1 may be effective in reducing the vascular risk associated with obesity.