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.

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.

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 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.

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.