CD36 Knockout Research Articles

CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis

Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.

What Do Experimental Models Teach Us About Comorbidities in Stroke?

There is a general consensus that animal models do not perfectly represent human conditions. This is not derived from any specific event, such as an ischemic episode, but rather reflects inherent differences in the physiology, anatomy, and metabolism among different species. Disparities among species also arise from differences in assessing functional recovery in stroke. Humans place a priority in regaining function in speech, cognition, and limb control, but these functions are not always readily translatable in animal models. Moreover, because functional recovery depends on the stroke type and location, induction of an ischemic lesion in a predefined area of an animal model, at best, reflects only a subtype of human stroke. Nevertheless, certain clinical pathophysiology features of stroke, such as acute outcomes and inflammatory/immune responses, are shared in animal models.1 Combined with complementary knowledge from in vitro studies, animal models can provide valuable insight into stroke pathology at a systems level. Analyses of translational inefficiencies in stroke model systems have led to the identification of underlying issues, which include, but are not limited to, rigor in experimental design, internal and external validity, negative bias, and insufficient statistical power. Together, these inefficiencies have steered collaborative international efforts in preclinical research to overcome these challenges.2 An additional issue of importance is the insufficient understanding of stroke pathophysiology beyond the acute phase and the primary reliance on infarct volume in reporting acute outcomes. Together with a lack of inclusion of comorbidities in animal models of stroke, the development of clinical protocols based on findings from normal metabolic animals may contribute to translational failures. In light of a recent concerted effort to address the future of translational stroke research,3 this review focuses on underlying translational issues and discusses insights gained from stroke models inclusive of comorbidities in the preclinical literature. ### Stroke Pathology: A Moving Target With Multiple Components Despite …

175 - LOS-1 Primarily Contributes to Pro-Inflammatory Macrophages Induced Foam Cell Formation

Modified LDL uptake by macrophages lead to formation of foam cells is an early event in atherosclerosis. Recent studies have suggested that macrophage polarization contributes to the progression of atherosclerosis. In this report, we investigated the hypothesis that macrophage polarization differentially affects modified LDL uptake and subsequent foam cell formation. Bone marrow-derived macrophages (BMDM) were exposed to LPS and IFNγ or IL-4 to induce M1 and M2 macrophage polarization. The formation of foam cells was assessed after exposing M1 and M2 macrophages to oxidized-LDL. Our results show that formation of foam cells was slightly increased in M2 macrophages. Interestingly, accumulation of neutral lipids was high in M1 macrophages indicating M1 polarization promotes foam cells. In M1 macrophages, mRNA and protein expression of scavenger receptor CD36 was lower compared to unpolarized macrophages. Notably, mRNA and protein expression of LOX-1 were increased several fold in M1 macrophages. M2 polarized BMDM from CD36 knock out or scavenger receptor-A (SR-A) knockout mice showed reduced foam cells. Surprisingly, M1 polarized macrophages from CD36 or SR-A knockout mice showed similar levels of foam cells compared to M1 polarized macrophages from wild type mice. These results suggest that CD36 and SR-A do not contribute to increased foam cells in M1 macrophages. Further studies also reveal that NF-kB activation through TLR4 is the major contributor for increased LOX1-expression and foam cell formation in M1 polarized macrophages. Macrophage plasticity, specifically polarization of macrophages to pro-inflammatory M1 phenotype promotes foam cell formation through LOX-1, which has implications for the progression of atherosclerotic plaques.

Cd36 knockout mice are protected against lithogenic diet-induced gallstones

The scavenger receptor and multiligand transporter CD36 functions to promote cellular free fatty acid uptake and regulates aspects of both hepatic and intestinal cholesterol metabolism. However, the role of CD36 in regulating canalicular and biliary cholesterol transport and secretion is unknown. Here, we show that germline Cd36 knockout (KO) mice are protected against lithogenic diet (LD)-induced gallstones compared with congenic (C57BL6/J) controls. Cd36 KO mice crossed into congenic L-Fabp KO mice (DKO mice) demonstrated protection against LD-induced gallstones, reversing the susceptibility phenotype observed in L-Fabp KO mice. DKO mice demonstrated reduced biliary cholesterol secretion and a shift into more hydrophophilic bile acid species, without changes in either BA pool size or fecal excretion. In addition, we found that the mean and maximum force of gallbladder contraction was increased in germline Cd36 KO mice, and gallbladder lipid content was reduced compared with wild-type controls. Finally, whereas germline Cd36 KO mice were protected against LD-induced gallstones, neither liver- nor intestine-specific Cd36 KO mice were protected. Taken together, our findings show that CD36 plays an important role in modifying gallstone susceptibility in mice, at least in part by altering biliary lipid composition, but also by promoting gallbladder contractility.

CD24 attenuates carbon tetrachloride-induced murine liver fibrosis

Objective To investigate the effects of CD24 on CCl4-induced murine liver fibrosis and to analyze the possible molecular mechanism. Methods Wild type (WT) and CD24 knockout (CD24-/-) C57BL/6 mice were treated with CCl4 through intraperitoneal injection. Levels of ALT in serum samples were detected and liver tissues were stained with hematoxylin and eosin (HE) to assess liver tissue injury. Sirius Red staining was used to observe liver fibrosis. Real-time PCR was performed to detect the expression of α-SMA (α-smooth muscle actin), Col1a1 (Collagen, typeⅠ, alpha 1), TGF-β1 (transforming growth factor-β1) and CD24 at mRNA level in liver tissues. Western blot was performed to analyze the expression of α-SMA and Col1a1 at protein level. Flow cytometry analysis was used to detect the macrophages in liver tissues. ELISA was used to detect the expression of TGF-β1 in the culture supernatants of M1 and M2 macrophages. Results The expression of CD24 at both mRNA and protein levels were up-regulated in mice with CCl4-induced liver fibrosis. HE staining showed that liver inflammation in CD24-/- mice was more severe than that in WT mice after treated with CCl4. Sirius Red staining of paraffin-embadded liver sections revealed that compared with WT mice, CD24-/- mice presented with more severe liver fibrosis. Moreover, α-SMA and Col1a1, indicators of liver fibrosis, in CD24-/- mice were significantly higher than those in WT mice. Flow cytometry analysis showed that murine hepatic macrophages significantly increased in CD24-/- mice than in WT mice following CCl4 treatment. Real-time PCR analysis also confirmed that significantly enhanced expression of TGF-β1 at mRNA level in liver tissues was observed in CD24-/- mice than in WT mice. TGF-β1 secreted in the culture supernatant of M2 macrophages derived from CD24-/- mice group was more than that of the WT mice group. No significant difference in TGF-β1 secretion in culture supernatant of M1 macrophages was observed between the two groups. Conclusion Taken together, these data suggest that CD24 plays an important role in attenuating CCl4-induced chronic inflammation and hepatic fibrosis in mice. The mechanism of CD24 in alleviating liver fibrosis might be through regulating intrahepatic macrophages, inhibiting the secretion of TGF-β1 by M2 macrophages and suppressing the activation of hepatic stellate cells. Key words: CD24; CCl4; Liver fibrosis; Macrophage; TGF-β1

Aorta macrophage inflammatory and epigenetic changes in a murine model of obstructive sleep apnea: Potential role of CD36

Obstructive sleep apnea (OSA) affects 8–10% of the population, is characterized by chronic intermittent hypoxia (CIH), and causally associates with cardiovascular morbidities. In CIH-exposed mice, closely mimicking the chronicity of human OSA, increased accumulation and proliferation of pro-inflammatory metabolic M1-like macrophages highly expressing CD36, emerged in aorta. Transcriptomic and MeDIP-seq approaches identified activation of pro-atherogenic pathways involving a complex interplay of histone modifications in functionally-relevant biological pathways, such as inflammation and oxidative stress in aorta macrophages. Discontinuation of CIH did not elicit significant improvements in aorta wall macrophage phenotype. However, CIH-induced aorta changes were absent in CD36 knockout mice, Our results provide mechanistic insights showing that CIH exposures during sleep in absence of concurrent pro-atherogenic settings (i.e., genetic propensity or dietary manipulation) lead to the recruitment of CD36(+)high macrophages to the aortic wall and trigger atherogenesis. Furthermore, long-term CIH-induced changes may not be reversible with usual OSA treatment.

Cardiomyocyte-specific ablation of CD36 accelerates the progression from compensated cardiac hypertrophy to heart failure.

Previous studies have shown that loss of CD36 protects the heart from dysfunction induced by pressure overload in the presence of diet-induced insulin resistance and/or obesity. The beneficial effects of CD36 ablation in this context are mediated by preventing excessive cardiac fatty acid (FA) entry and reducing lipotoxic injury. However, whether or not the loss of CD36 can prevent pressure overload-induced cardiac dysfunction in the absence of chronic exposure to high circulating FAs is presently unknown. To address this, we utilized a tamoxifen-inducible cardiomyocyte-specific CD36 knockout (icCD36KO) mouse and genetically deleted CD36 in adulthood. Control mice (CD36 floxed/floxed mice) and icCD36KO mice were treated with tamoxifen and subsequently subjected to transverse aortic constriction (TAC) surgery to generate pressure overload-induced cardiac hypertrophy. Consistent with CD36 mediating a significant proportion of FA entry into the cardiomyocyte and subsequent FA utilization for ATP production, hearts from icCD36KO mice were metabolically inefficient and displayed signs of energetic stress, including activation of the energetic stress kinase, AMPK. In addition, impaired energetics in icCD36KO mice contributed to a rapid progression from compensated hypertrophy to heart failure. However, icCD36KO mice fed a medium-chain FA diet, whereby medium-chain FAs can enter into the cardiomyocyte independent from CD36, were protected from TAC-induced heart failure. Together these data suggest that limiting FA uptake and partial inhibition of FA oxidation in the heart via CD36 ablation may be detrimental for the compensated hypertrophic heart in the absence of sufficiently elevated circulating FAs to provide an adequate energy source.NEW & NOTEWORTHY Limiting CD36-mediated fatty acid uptake in the setting of obesity and/or insulin resistance protects the heart from cardiac hypertrophy and dysfunction. However, cardiomyocyte-specific CD36 ablation in the absence of elevated circulating fatty acid levels accelerates the progression of pressure overload-induced cardiac hypertrophy to systolic heart failure.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein A-I-mimetic peptide 5A antagonizes CD36 invitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild-type to CD36 knockout mice and wild-type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild-type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild-type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreased renal expression of inflammasome genes. Thus,CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression.

CD24 Induces the Activation of β-Catenin in Intestinal Tumorigenesis

Background: CD24 is a glycosylphosphatidylinositol-linked protein that functions as an adhesion molecule and is overexpressed at an early stage of CRC. The Wnt/β-catenin signaling pathway plays an important role in the CRC carcinogenesis process. We have previously shown that CD24 could affect the tumorigenesis process in ApcMin/+ mice. Methods: CD24-inducible 293T-RExTM cells previously developed in our lab, HT29 and SW480 CRC cells were used to study this interaction in vitro. ApcMin/+ and CD24 knockout (KO) mice, both on a C57BL/6J genetic background, were crossed to generate double KO transgenic mice. Large bowel polyps were counted macroscopically and small bowel polyps were verified microscopically. Results: In vitro Western blotting analyses showed that induced expression of CD24 led to the activation of β-catenin. Co-immunoprecipitation studies of CD24 and β-catenin indicated that these two proteins might be interacting. Cytoplasmic/nuclear fractionation showed that more active β-catenin enters the nucleus in cells that express CD24 compared to control cells. In addition, TOP/FOP luciferase reporter assay showed a significant increase in luciferase activity upon CD24 expression induction.Conversely, down-regulation of CD24 by mAbs and siRNA caused a decrease in the levels of active β-catenin. Depletion of CD24 alleles in ApcMin/+ mice led to a significant reduction in the number of polyps in the small and large intestine. The ApcMin/+mice displayed severe splenomegaly compared to ApcMin/+/CD24+/- mice and double KO mice were similar to WT mice with normal spleen size. HGB and RBC levels were significantly lower than in the double KO mice. Conclusions: CD24 plays a major role in intestinal tumorigenesis. It interacts with the Wnt pathway by activating β-catenin. Down regulation of CD24 reduces the polyp burden in mice and therefore it might serve as an important target in the therapy of CRC.

Behavioral characterization of CD36 knockout mice with SHIRPA primary screen

CD36 is a member of the class B scavenger receptor family of cell surface proteins, which plays a major role in fatty acid, glucose and lipid metabolism. Besides, CD36 functions as a microglial surface receptor for amyloid beta peptide. Regarding this, we suggest CD36 might also contribute to neuropsychiatric disease. The aim of this study was to achieve a behavioral phenotype of CD36 knockout (CD36−/−) mice. We characterized the behavior of CD36−/− mice and C57BL/6J mice by subjecting them to a series of tests, which include SHIRPA primary behavioral screen test, 1% sucrose preference test, elevated plus-maze test, open-field test and forced swimming test. The results showed that CD36−/− mice traversed more squares, emitted more defecation, exhibited higher tail elevation and had more aggressive behaviors than C57BL/6J mice. The CD36−/− mice spent more time and traveled longer distance in periphery zone in the open-field test. Meanwhile, the numbers that CD36−/− mice entered in the open arms of elevated plus-maze were reduced. These findings suggest that CD36−/− mice present an anxious phenotype and might be involved in neuropsychiatric disorders.

Myocardial energy provision is preserved by increased utilization of glucose and ketone bodies in CD36 knockout mice

AimsCD36 is an important transporter of long-chain fatty acids (LCFAs) in the myocardium. As we have reported previously, CD36-deficient patients demonstrate a marked reduction in myocardial uptake of 123I-15-(p-iodophenyl)-(R, S)-methyl pentadecanoic acid (BMIPP), which is an analog of LCFAs, while myocardial 18F-fluorodeoxy-glucose (FDG) uptake is increased. However, it has not been clarified whether energy provision is preserved in patients with CD36 deficiency. The aims of the current study were to investigate the myocardial uptake of glucose and alterations in myocardial metabolites in wild-type (WT) and CD36 knockout (KO) mice. Methods and resultsHigh-resolution positron emission tomography (PET) demonstrated markedly enhanced glucose uptake in KO mouse hearts compared with those of WT mice in real-time. The myocardial protein expression of glucose transporter protein 1 (GLUT1) was significantly enhanced in KO mice compared to WT mice, whereas that of GLUT4 was not altered. While the myocardial expression of genes involved in fatty acid metabolism did not increase in KO mice, that of genes related to glucose utilization compensatorily increased in KO mice. The metabolomic analysis of cardiac tissues revealed that the myocardial concentrations of ATP and phosphocreatine were maintained, even in KO mice. The concentration of 3-hydroxybutyric acid and mRNA expression of hydroxybutyrate dehydrogenase in the heart were significantly higher in KO than in WT mice. ConclusionThese data suggest that high-energy phosphate might be preserved by the increased utilization of glucose and ketone bodies in CD36KO mouse hearts under conditions of deficient LCFA uptake.

Su1113 In Search of a Non-Invasive Blood Test for Diagnosis and Monitoring of EoE: A Prospective Study of a Serum Biomarker Panel

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches

Su1887 Increased Expression of PPARγ in CD24 Knockout (KO) Mice and Gender-Specific Metabolic Changes: A Unique Model of Insulin-Sensitive Obesity

Su1887 Increased Expression of PPARγ in CD24 Knockout (KO) Mice and Gender-Specific Metabolic Changes: A Unique Model of Insulin-Sensitive Obesity

Su1112 A Set of Clinical and Endoscopic Features Predicts the Presence of Eosinophilic Esophagitis With High Accuracy, Allowing Omission of Low-Yield Biopsies: Results From a Prospective Study

increasing in the population especially in pediatrics and reach 1:1000 in diseases such as EoE (Eosinophilic Esophagitis) (Lee 2013). CD24 is a small, mucin-like glycoprotein that functions both in signal transduction and as an adhesion molecule (Sagiv, 2008). CD24 is expressed on many hematopoietic cells, non-hematopoietic cells and malignancies (Fang, 2010). Previous studies in eosinophils have suggested CD24 as an IL-5-upregulated gene that is involved in eosinophil apoptosis and survival (Temple, 2011). The role evoked for CD24 in IL-5-induced survival of eosinophils remains to be investigated. We hypothesize that CD24 is a key molecular checkpoint, regulating eosinophil functions in chronic mucosal GI diseases, all of which are characterized by significant eosinophil infiltration. Aim: 1) To assess the expression of CD24 in eosinophils in the GI tract in wild-type (WT) mice; 2) To assess the level of eosinophils in the GI tract in CD24 knockout (-/-) and to compare it to WT mice.Methods: We have previously established a colony of CD24-/mice, on a C57BL/ 6J genetic background, and their WT littermates at our local animal facility. The levels of eosinophils were assessed in WT and CD24-/mice. Bone marrow (BM) cells were collected, and grown in medium containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5). On day 13 the cells were counted and levels of eosinophils were assessed by FACS. The levels of eosinophil and CD24 expression in different tissues were also evaluated. Results: Eosinophil counts in BM extracted from mice were significantly (p-value=0.0436) higher in CD24-/compared to WT mice, (Fig. 1). Fluorescense intensity was stronger in CD24-/compared to WT mice even though there were no differences in the eosinophil population of mature cells (Fig 2). Eosinophil count was higher in BM from CD24-/compared to WT mice, and lower in blood and addipose tissue (Fig. 3). CD24 expression was higher in peripheral tissues compared to the blood and BM (Fig 4). Conclusion: Based on our preliminary results, it is suggested that CD24 has an important role in negatively regulating eosinophil proliferation/maturation/chemotaxis. Understanding the role of eosinophil in GI disorders may have a large imapct on our understanding of the EGIDs. Targeting specific proteins that play an important role in these processes may lead to novel therapeutic approaches

Siglec-G–CD24 axis controls the severity of graft-versus-host disease in mice

AbstractActivation of sialic-acid–binding immunoglobulin-like lectin-G (Siglec-G) by noninfectious damage-associated molecular patterns controls innate immune responses. However, whether it also regulates T-cell–mediated adaptive immune responses is not known. Graft-versus-host reaction is a robust adaptive immune response caused by allogeneic hematopoietic cell transplantation that have been activated by antigen-presenting cells (APCs) in the context of damaged host tissues following allogeneic hematopoietic cell transplantation. The role of infectious and noninfectious pattern recognition receptor–mediated activation in the induction and aggravation of graft-versus-host disease (GVHD) is being increasingly appreciated. But the role of pathways that control innate immune responses to noninfectious stimuli in modulating GVHD has heretofore not been recognized. We report that Siglec-G expression on host APCs, specifically on hematopoietic cells, negatively regulates GVHD in multiple clinically relevant murine models. Mechanistic studies with various relevant Siglec-G and CD24 knockout mice and chimeric animals, along with rescue experiments with novel CD24 fusion protein demonstrate that enhancing the interaction between Siglec-G on host APCs with CD24 on donor T cells attenuates GVHD. Taken together, our data demonstrate that Siglec-G–CD24 axis, controls the severity of GVHD and suggest that enhancing this interaction may represent a novel strategy for mitigating GVHD.

Mo1775 High Expression Level of PPARγ in CD24 Knockout Mice and Gender Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity

Mo1775 High Expression Level of PPARγ in CD24 Knockout Mice and Gender Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APCMin/CD24 double knockout transgenic mice

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APC(Min/+) mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation-associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β-catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Low-Bone-Mass Phenotype of Deficient Mice for the Cluster of Differentiation 36 (CD36)

Bone tissue is continuously remodeled by bone cells and maintenance of its mass relies on the balance between the processes of resorption and formation. We have reported the expression of numerous scavenger receptors, namely scavenger receptor (SR) class B type I and II (SR-BI and SR-BII), and CD36, in bone-forming osteoblasts but their physiological roles in bone metabolism are still unknown. To unravel the role of CD36 in bone metabolism, we determined the bone phenotype of CD36 knockout (CD36KO) mice and characterized the cell functions of osteoblasts lacking CD36. Weights of CD36KO mice were significantly lower than corresponding wild-type (WT) mice, yet no significant difference was found in femoral nor tibial length between CD36KO and WT mice. Analysis of bone architecture by micro-computed tomography revealed a low bone mass phenotype in CD36KO mice of both genders. Femoral trabecular bone from 1 to 6 month-old CD36KO mice showed lower bone volume, higher trabecular separation and reduced trabeculae number compared to WT mice; similar alterations were noticed for lumbar vertebrae. Plasma levels of osteocalcin (OCN) and N-terminal propeptide of type I procollagen (PINP), two known markers of bone formation, were significantly lower in CD36KO mice than in WT mice, whereas plasma levels of bone resorption markers were similar. Accordingly, histology highlighted lower osteoblast perimeter and reduced bone formation rate. In vitro functional characterization of bone marrow stromal cells and osteoblasts isolated from CD36KO mice showed reduced cell culture expansion and survival, lower gene expression of osteoblastic Runt-related transcription factor 2 (Runx2) and osterix (Osx), as well as bone sialoprotein (BSP) and osteocalcin (OCN). Our results indicate that CD36 is mandatory for adequate bone metabolism, playing a role in osteoblast functions ensuring adequate bone formation.

Cardiomyocyte-specific ablation of CD36 improves post-ischemic functional recovery

Although pre-clinical evidence has suggested that partial inhibition of myocardial fatty acid oxidation (FAO) and subsequent switch to greater glucose oxidation for ATP production can prevent ischemia/reperfusion injury, controversy about this approach persists. For example, mice with germline deletion of the FA transporter CD36, exhibited either impaired or unchanged post-ischemic functional recovery despite a 40–60% reduction in FAO rates. Because there are limitations to cardiac studies utilizing whole body CD36 knockout (totalCD36KO) mice, we have now generated an inducible and cardiomyocyte-specific CD36 KO (icCD36KO) mouse to better address the role of cardiomyocyte CD36 and its regulation of FAO and post-ischemic functional recovery. Four to six weeks following CD36 ablation, hearts from icCD36KO mice had significantly decreased FA uptake compared to controls, which was paralleled by significant reductions in intramyocardial triacylglycerol content. Analysis of cardiac energy metabolism using ex vivo working heart perfusions showed that reduced FAO rates were compensated by enhanced glucose oxidation in the hearts from icCD36KO mice. In contrast to the totalCD36KO mice, hearts from icCD36KO mice exhibited significantly improved functional recovery following ischemia/reperfusion (18min of global no-flow ischemia followed by 40min of aerobic reperfusion). This improved recovery was associated with lower calculated proton production prior to and following ischemia compared to controls. Moreover, the amount of ATP generated relative to cardiac work was significantly lower in the hearts from icCD36KO mice compared to controls, indicating significantly increased cardiac efficiency in the hearts from icCD36KO mice. These data provide genetic evidence that reduced FAO as a result of diminished CD36-mediated FA uptake improves post-ischemic cardiac efficiency and functional recovery. As such, targeting cardiomyocyte FA uptake and FAO via inhibition of CD36 in the adult myocardium may provide therapeutic benefit during ischemia–reperfusion.