Kristy Ou

Knockout of Adamts7, a Novel Coronary Artery Disease Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Modulating platelet reactivity through control of RGS18 availability

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

Knockout of Adamts7, A Novel CAD Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Overexpression of ST5, an activator of Ras, has no effect on β-cell proliferation in adult mice

Objective Both Type I and Type II diabetes mellitus result from insufficient functional β-cell mass. Efforts to increase β-cell proliferation as a means to restore β-cell mass have been met with limited success. Suppression of Tumorigenicity 5 (ST5) activates Ras/Erk signaling in the presence of Epidermal Growth Factor (EGF). In the pancreatic islet, Ras/Erk signaling is required for augmented β-cell proliferation during pregnancy, suggesting that ST5 is an appealing candidate to enhance adult β-cell proliferation. We aimed to test the hypothesis that overexpression of ST5 drives adult β-cell proliferation. Methods We utilized a doxycycline-inducible bitransgenic mouse model to activate β-cell-specific expression of human ST5 in adult mice at will. Islet morphology, β-cell proliferation, and β-cell mass in control and ST5-overexpressing (ST5 OE) animals were analyzed by immunofluorescent staining, under basal and two stimulated metabolic states: pregnancy and streptozotocin (STZ)-induced β-cell loss. Results Doxycycline treatment resulted in robust ST5 overexpression in islets from 12-16 week-old ST5 OE animals compared to controls, without affecting the islet morphology and identity of the β-cells. Under both basal and metabolically stimulated pregnancy states, β-cell proliferation and mass were comparable in ST5 OE and control animals. Furthermore, there was no detectable difference in β-cell proliferation between ST5 OE and control animals in response to STZ-induced β-cell loss. Conclusions We successfully derived an inducible bitransgenic mouse model to overexpress ST5 specifically in β-cells. However, our findings demonstrate that ST5 overexpression by itself has no mitogenic effect on the adult β-cell under basal and metabolically challenged states.

Targeted demethylation at the CDKN1C/p57 induces human β cell replication

The loss of insulin-secreting &bgr; cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of &bgr; cells for transplantation remains a challenge because adult &bgr; cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human &bgr; cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive &bgr; cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator–like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased &bgr; cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing &bgr; cell proliferation, which may one day alleviate the scarcity of transplantable &bgr; cells for the treatment of diabetes.

The Dysregulation of the DLK1-MEG3 Locus in Islets From Type 2 Diabetics is Mimicked by Targeted Epimutation of its Promoter with TALE-DNMT Constructs

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.

Knockout of Adamts7, a Novel Coronary Artery Disease Locus in Humans, Reduces Atherosclerosis in MiceCLINICAL PERSPECTIVE

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.