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Obesity is becoming a global epidemic, predisposing to Type 2 diabetes, cardiovascular disease, fatty liver disease, pulmonary disease, osteoarthritis and cancer. Therefore, understanding the biology of adipocyte expansion in response to overnutrition is critical to devising strategies to treat obesity, and the associated burden of morbidity and mortality. Through exploratory patch-clamp experiments in freshly isolated primary murine and human adipocytes, we recently determined that SWELL1/LRRC8a, a leucine-rich repeat containing transmembrane protein, functionally encoded an ion channel signalling complex (the volume-regulated anion channel, or VRAC) on the adipocyte plasma membrane. The SWELL1-/LRRC8 channel complex activates in response to increases in adipocyte volume and in the context of obesity. SWELL1 is also required for insulin-PI3K-AKT2 signalling to regulate adipocyte growth and systemic glycaemia. This commentary delves further into our working models for the molecular mechanisms of adipocyte SWELL1-mediated VRAC activation, proposed signal transduction mechanisms, and putative impact on adipocyte hypertrophy during caloric excess.

Type 2 diabetes (T2D) is associated with insulin resistance, impaired insulin secretion from the pancreatic β-cell, and nonalcoholic fatty liver disease (NAFLD). SWELL1 (LRRC8a) ablation impairs adipose and skeletal muscle insulin-pAKT2 signaling, β-cell insulin secretion and glycemic control - suggesting that SWELL1-LRRC8 complex dysfunction contributes to T2D pathogenesis. Here, we show that ICl,SWELLand SWELL1 protein are reduced in adipose and β-cells in murine and human T2D. Combining cryo-electron microscopy, molecular docking, medicinal chemistry, and functional studies, we define a structure activity relationship to rationally-designed active derivatives (SN-40X) of a SWELL1 channel inhibitor (DCPIB/SN-401), that bind the SWELL1-LRRC8 hexameric complex, restore SWELL1-LRRC8 protein, plasma membrane trafficking, signaling and islet insulin secretion via SWELL1-dependent mechanisms.In vivo, SN-401 and active SN-40X compounds restore glycemic control and prevents NAFLD by improving insulin-sensitivity and insulin secretion in murine T2D. These findings demonstrate that small molecule SWELL1 modulators restore SWELL1-dependent insulin-sensitivity and insulin secretion in T2D and may represent a first-in-class therapeutic approach for T2D and NAFLD.

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism; however, the molecular mechanosensor remains unknown. Here, we show that SWELL1 (Lrrc8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle-targeted Lrrc8a KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to wild-type (WT) mice. These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo., eLife digest Skeletal muscles – the force-generating tissue attached to bones – must maintain their mass and health for the body to work properly. It is therefore necessary to understand how an organism can regulate the way skeletal muscles form, grow and heal. A multitude of factors can control how muscles form, including mechanical signals. The molecules that can sense these mechanical stimuli, however, remain unknown. Mechanoresponsive ion channels provide possible candidates for these molecular sensors. These proteins are studded through the cell membranes, where they can respond to mechanical changes by opening and allowing the flow of ions in and out of a cell, or by changing interactions with other proteins. The SWELL1 protein is a component of an ion channel known as VRAC, which potentially responds to mechanical stimuli. This channel is associated with many biological processes such as cells multiplying, migrating, growing and dying, but it is still unclear how. Here, Kumar et al. first tested whether SWELL1 controls how skeletal muscle precursors mature into their differentiated and functional form. These experiments showed that SWELL1 regulates this differentiation process under the influence of the hormone insulin, as well as mechanical signals such as cell stretching. In addition, this work revealed that SWELL1 relies on an adaptor molecule called GRB2 to relay these signals in the cell. Next, Kumar et al. genetically engineered mice lacking SWELL1 only in skeletal muscle. These animals had smaller muscle cells, as well as muscles that were weaker and less enduring. When raised on a high-calorie diet, the mutant mice also got more obese and developed resistance to insulin, which is an important step driving obesity-induced diabetes. Together, these findings show that SWELL1 helps to regulate the formation and function of muscle cells, and highlights how an ion channel participates in these processes. Healthy muscles are key for overall wellbeing, as they also protect against obesity and obesity-related conditions such as type 2 diabetes or nonalcoholic fatty liver disease. This suggests that targeting SWELL1 could prove advantageous in a clinical setting.

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (LRRC8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. SWELL1 over-expression in SWELL1 KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted SWELL1 KO mice have smaller myofibers, generate less forceex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the SWELL1-LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiationin vitroand skeletal myofiber size, muscle function, adiposity and systemic metabolismin vivo.

Insulin secretion is initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC requires β-cell membrane depolarization, which is regulated by a balance of depolarizing and hyperpolarizing ionic currents. Here, we show that SWELL1 mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in both murine and human β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this contributes to membrane depolarization and activation of VGCC-dependent intracellular calcium signaling. SWELL1 depletion in MIN6 cells and islets significantly impairs glucose-stimulated insulin secretion. Tamoxifen-inducible β-cell-targeted Swell1 KO mice have normal fasting serum glucose and insulin levels but impaired glucose-stimulated insulin secretion and glucose tolerance; and this is further exacerbated in mild obesity. Our results reveal that β-cell SWELL1 modulates insulin secretion and systemic glycaemia by linking glucose-mediated β-cell swelling to membrane depolarization and activation of VGCC-triggered calcium signaling., Insulin secretion by β-cells is stimulated by glucose and is dependent on the induction of β-cell membrane depolarization, mainly driven by the closure of KATP channels, which in turn promotes voltage-gated Ca2+ channel opening. Here Kang et al. show that the volume-regulated anion channel, SWELL1, is involved in glucose-stimulated calcium increase and insulin secretion.

Objective Reduction of oxidized methionines is emerging as a major protein repair pathway. The lack of methionine sulfoxide reductase A (MsrA) exacerbates cardiovascular disease phenotypes driven by increased oxidative stress. However, the role of MsrA on maintaining cellular homeostasis in the absence of excessive oxidative stress is less well understood. Methods and results Constitutive genetic deletion of MsrA increased formation of p62-containing protein aggregates, activated autophagy, and decreased a marker of apoptosis in vascular smooth muscle cells (VSMC). The association of Keap1 with p62 was augmented in MsrA-/- VSMC. Keap1 targets the transcription factor Nrf2, which regulates antioxidant genes, for proteasomal degradation. However, in MsrA-/- VSMC, the association of Nrf2 with Keap1 was diminished. Whereas Nrf2 mRNA levels were not decreased in MsrA-/- VSMC, we detected decreased ubiquitination of Nrf2 and a corresponding increase in total Nrf2 protein in the absence of biochemical markers of oxidative stress. Moreover, nuclear-localized Nrf2 was increased under MsrA deficiency, resulting in upregulation of Nrf2-dependent transcriptional activity. Consequently, transcription, protein levels and enzymatic activity of glutamate-cysteine ligase and glutathione reductase were greatly augmented in MsrA-/- VSMC. Summary Our findings demonstrate that reversal of methionine oxidation is required for maintenance of cellular homeostasis in the absence of increased oxidative stress. These data provide the first link between autophagy and activation of Nrf2 in the setting of MsrA deletion., Graphical abstract fx1, Highlights • With MsrA deletion, p62-containing intracellular protein aggregates are increased. • Autophagy is increased in proliferating MsrA-/- smooth muscle cells. • Increased association of Keap-1 with p62 derepresses Nrf2 in MsrA-/- cells. • Nrf2 activation in MsrA-/- cells occurs without increased oxidative stress.

Obesity is associated with a loss of insulin-sensitivity and systemic dysglycemia, resulting in Type 2 diabetes, however the molecular mechanisms underlying this association are unclear. Through adipocyte patch-clamp studies, we recently showed that SWELL1 is required for the Volume-Regulated Anion Current (VRAC) in adipocytes and that SWELL1-mediated VRAC is activated by both mechanical and pathophysiological adipocyte expansion. We also demonstrated that adipocyte SWELL1 is required for maintaining insulin signaling and glucose homeostasis, particularly in the setting of obesity. Here we show that SWELL1 protein expression is induced in subcutaneous fat, visceral fat and liver in the setting of obesity. Long- term AAV/rec2-shRNA mediated SWELL1 knock-down in both fat and liver are associated with increased weight gain, increased adiposity and exacerbated insulin resistance in mice raised on a high-fat diet. These data further support the notion that SWELL1 induction occurs in insulin- sensitive tissues (liver and adipose) in the setting of over-nutrition and contributes to improved systemic glycemia by supporting enhanced insulin-sensitivity.

Insulin secretion from the pancreatic β-cell initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC depends on the β-cell membrane potential, which is in turn mediated by the balance of depolarizing (excitatory) and hyperpolarizing (inhibitory) ionic currents1-3. While much attention has focused on inhibitory potassium currents4-10 there is little knowledge about the excitatory currents required to depolarize the β-cell, including the molecular identity of these excitatory currents3. Here we show that SWELL1 (LRRC8a) mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this is required for both glucose-stimulated and hypotonic swell-mediated activation of VGCC-dependent intracellular calcium signaling in β-cells. SWELL1 KO MIN6 cells and β-cell targeted SWELL1 KO murine islets exhibit significantly impaired glucose-stimulated insulin secretion, with preserved insulin content in vitro. Tamoxifen-inducible β-cell targeted SWELL1 KO mice have normal fasting insulin levels but display markedly impaired glucose-stimulated insulin secretion. Our results reveal a physiological role for SWELL1 as a glucose sensor - linking glucose-mediated β-cell swelling to SWELL1-dependent activation of VGCC-triggered calcium signaling, and highlights SWELL1-mediated “swell-secretion” coupling as required for glucose-stimulated insulin secretion.

The patch-clamp technique allows for the study of ion channel activity in the native adipocyte environment to better understand the contributions of ion channels to adipocyte signaling. Here, we describe methods for isolating primary mature adipocytes from both mouse and human white adipose tissues (subcutaneous and visceral). From the same preparation, we describe how to culture and differentiate preadipocytes isolated from the stromal vascular fraction. We then describe in detail patch-clamp methods, including both whole-cell and perforated-patch configurations.

Adipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signalling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine-Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signalling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signalling. In vivo, shRNA-mediated SWELL1 knockdown and adipose-targeted SWELL1 knockout reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.

This paper studies the linear stability and dynamic structure for a generalized non-linear heat equation, and obtains novel analytic solutions such as homoclinc orbit and breather solitary solutions for the first time based on Hirota method.

We mapped 226 unique integration sites in human hepatoma cells following gene transfer with a feline immunodeficiency virus (FIV)-based lentivirus vector. FIV integrated across the entire length of the transcriptional units. Microarray data indicated that FIV integration favored actively transcribed genes. Approximately 21% of FIV integrations within transcriptional units occurred in genes regulated by the LEDGF/p75 transcriptional coactivator. DNA in regions of FIV insertion sites exhibited a “bendable” structure and a pattern of duplex destabilization favoring strand separation. FIV integration preferences are more similar to those of primate lentiviruses and distinct from those of Moloney murine leukemia virus, avian sarcoma leukosis virus, and foamy virus.

SUMMARY Adipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signaling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signaling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signaling. In vivo, shRNA-mediated SWELL1 knock-down and adipose-targeted SWELL1 knock-out reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin-sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.

Introduction: Neointima formation is the leading cause of restenosis after balloon angioplasty and surgical endarterectomy. ROS production promotes neointimal hyperplasia and causes methionine oxidation. Recent data suggest that oxidation of methionines can alter specific protein activity and induce cell signaling. Methionine Sulfoxide Reductase A (MsrA) reverses methionine oxidation. In humans, two independent genome-wide association studies have found MsrA polymorphisms to correlate with increased ischemic cardiovascular disease. Hypothesis: MsrA protects against neointimal formation. Methods: Immunofluorescence for MsrA was performed in autopsy specimen of arteries with and without neointimal hyperplasia. 12-week old WT and MsrA-/- mice underwent ligation of the left common carotid artery and received two injections of BrdU prior to sacrifice. Fourteen days after ligation, the neointimal area was determined using Image J. Cell proliferation and apoptosis in the neointima were quantified by BrdU and TUNEL staining. Proliferation of vascular smooth muscle cells (VSMC) from MsrA-/- and WT mice was determined by cell counts and FACS analysis. Levels of cell cycle proteins, pAkt, pGSK3β were analyzed by immunoblot in MsrA-/- and WT VSMC, mRNA levels by qrtPCR. Results: MsrA was detected in all layers of the vascular wall and in neointima in humans. Neointimal area was significantly increased in MsrA-/- compared to WT mice 14 days post-ligation (n=5-9, p Conclusion: These data indicate that MsrA regulates neointima formation after vascular injury and support a role for MsrA in the regulation of Akt signaling.

Activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and reactive oxygen species (ROS) promote neointimal hyperplasia after vascular injury. CaMKII can be directly activated by ROS through oxidation. In this study, we determined whether abolishing the oxidative activation site of CaMKII alters vascular smooth muscle cell (VCMC) proliferation, migration and apoptosis in vitro and neointimal formation in vivo. VSMC isolated from a knock-in mouse with oxidation-resistant CaMKIIδ (CaMKII M2V) displayed similar proliferation but decreased migration and apoptosis. Surprisingly, ROS production and expression of the NADPH oxidase subunits p47 and p22 were decreased in M2V VSMC, whereas superoxide dismutase 2 protein expression was upregulated. In vivo, after carotid artery ligation, no differences in neointimal size or remodeling were observed. In contrast to VSMC, CaMKII expression and autonomous activity were significantly higher in M2V compared to WT carotid arteries, suggesting that an autoregulatory mechanism determines CaMKII activity in vivo. Our findings demonstrate that preventing oxidative activation of CaMKII decreases migration and apoptosis in vitro and suggest that CaMKII regulates ROS production. Our study presents novel evidence that CaMKII expression in vivo is regulated by a negative feedback loop following oxidative activation.

Objective Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. Methods and Results Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling. Conclusion CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

The multifunctional CaMKII has been implicated in vascular smooth muscle cell (VSMC) migration, but little is known regarding its downstream targets that mediate migration. Here, we examined whether CaMKII regulates migration through modulation of matrix metalloproteinase 9 (MMP9). Using CaMKIIδ−/−mice as a model system, we evaluated migration and MMP9 regulation in vitro and in vivo. After ligation of the common carotid artery, CaMKII was activated in the neointima as determined by oxidation and autophosphorylation. We found that MMP9 was robustly expressed in the neointima and adventitia of carotid-ligated wild-type (WT) mice but was barely detectable in CaMKIIδ−/−mice. The perimeter of the external elastic lamina, a correlate of migration-related outward remodeling, was increased in WT but not in CaMKIIδ−/−mice. Migration induced by serum, platelet-derived growth factor, and tumor necrosis factor-α (TNF-α) was significantly decreased in CaMKIIδ−/−as compared with WT VSMCs, but migration was rescued with adenoviral overexpression of MMP9 in CaMKIIδ−/−VSMCs. Likewise, overexpression of CaMKIIδ in CaMKIIδ−/−VSMCs increased migration, whereas an oxidation-resistant mutant of CaMKIIδ did not. TNF-α strongly induced CaMKII oxidation and autophosphorylation as well as MMP9 activity, mRNA, and protein levels in WT, but not in CaMKIIδ−/−VSMC. Surprisingly, TNF-α strongly induced MMP9 promoter activity in WT and CaMKIIδ−/−VSMC. However, the MMP9 mRNA stability was significantly decreased in CaMKIIδ−/−VSMC. Our data demonstrate that CaMKII promotes VSMC migration through posttranscriptional regulation of MMP9 and suggest that CaMKII effects on MMP9 expression may be a therapeutic pathway in vascular injury.

Background The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. Methods We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. Results In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. Conclusions These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. Abbreviations Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

Top of pageAbstract Hemophilia A is an X-linked genetic disorder disease resulting in quantitative or qualitative deficiencies in coagulation Factor VIII and the risk for sustained bleeding after trauma or injury. The disorder is an attractive candidate for treatment by gene transfer because only partial correction is required for clinically relevant effects. We are investigating the Sleeping Beauty transposon vector system to express FVIII, using a liver-specific mouse albumin enhancer and human alpha-1-antitrypsin promoter (Alb/HAAT) to direct human FVIII cDNA expression. Transgenes under investigation include B-domain deleted human FVIII (BDD-FVIII) and a FVIII cDNA engineered to include several asparagine-linked oligosaccharides within a short B-domain spacer (F226aa/N6, Pipe et al). Using E16 hemophilia A mice on a congenic C57/BL6 background, we are contrasting expression from these constructs following hydrodynamic tail vein injection. Transposon pT3-Alb/HAAT-BDD-FVIII resulted in plasma FVIII levels of 14 ng/ml 1 week post-injection which gradually declined to |[sim]|4 ng/ml by 3 months. Preliminary results with transposon pT3-Alb/HAAT-F226aa/N6 demonstrate plasma FVIII levels of |[sim]|15 ng/ml 1 month post-injection. No antibodies to hFVIII were detected in either group. These results indicate that a Sleeping Beauty transposon with a liver-specific promoter can direct human factor VIII expression in a mouse model of FVIII and attain partial correction of the deficient protein.

Hemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)–based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human α1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain–deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti–human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

A decline in stress tolerance is a hallmark of aging. For instance, older organisms showed extensive hepatic damage, along with increased morbidity and mortality, after environmental heating. We hypothesized that hyperthermic challenge would produce exaggerated oxidative stress in old animals, leading to increased hepatic injury. After a heat-stress protocol, time-course changes in reactive oxygen species (ROS) levels, oxidative damage markers, glutathione (GSH)/glutathione disulfide (GSSG) ratios, and activation of stress-response transcription factors (AP-1 and NF-kappaB) were measured in young and old rats. A small, transient increase in hepatic oxidative damage, with minimal injury, was observed in young rats. However, old rats showed widespread hepatic injury that was manifested over a 24 h period after heating. This pathology was preceded by elevated steady-state levels of ROS, along with large increases in lipid peroxidation products, prolonged hepatic DNA oxidation damage, aberrant GSH/GSSG profiles, and altered activation patterns for AP-1. These data indicate that young animals have an effective oxidation-reduction buffering system in the liver that provides protection from oxidative damage to intracellular macromolecules under stress conditions. In sharp contrast, an environmental challenge in older animals produces exaggerated oxidative stress and alterations in signal transduction pathways, which can contribute to cellular dysfunction and age-related reductions in stress tolerance.

Objective Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis. Methods and Results Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling. Conclusion CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Hemophilia A is an X-linked genetic disorder resulting in deficiencies in coagulation Factor VIII and a risk of sustained bleeding after trauma or injury. Hemophilia A is an attractive candidate for treatment by gene transfer because only partial correction is required for clinically relevant effects. We previously reported that gene transfer to the liver of E16 FVIII deficient mice using a lentiviral vector derived from feline immunodeficiency virus (FIV) resulted in sustained partial correction of the bleeding disorder. We are now investigating the impact of several vector and transgene modifications on gene transfer to hepatocytes using baculovirus GP64 pseudotyped FIV. The goals of the project are to improve transgene expression through several vector modifications and to test the optimized vector's effects on protein expression. Modifications include rendering the lentiviral vector |[ldquo]|self-inactivating|[rdquo]| by deleting the U3 region of the 3’ LTR, inserting the FIV central polypurine tract (cPPT) element upstream of the internal promoter, and incorporating the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) downstream of a luciferase reporter gene or the FVIII cDNA. Furthermore, two sites were mutated, a major splice donor and the start codon of the partial Gag sequence, preventing production of undesired product. In addition, the post- transcriptional control element from the 5’ LTR of spleen necrosis virus (SNVU5) was incorporated into the delivery vector. The liver- specific mouse albumin enhancer and human alpha-1-antitrypsin promoter (Alb/HAAT) are being used to direct firefly luciferase, B- domain deleted human FVIII (BDD-FVIII), and a FVIII cDNA engineered to include several asparagine-linked oligosaccharides within a short B-domain spacer (F226aa/N6) in wild type and E16 FVIII deficient mice on a congenic C57/BL6 background. Findings from these ongoing experiments will be reported.

Top of pageAbstract Hemophilia A is a common X-linked coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudotyped FIV efficiently and showed excellent hepatocyte tropism. The GP64 pseudotyped FIV was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human 1-antitrypsin promoter), further enhanced transgene expression in hepatoctyes. We generated a GP64-pseudotyped FIV vector encoding the B domain-deleted human FVIII coding region driven by the liver-specific promoter, with two beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti-human FVIII antibodies, and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64 pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

Retroviral vector-mediated gene transfer holds promise in the treatment of a variety of genetic diseases. A distinguishing feature of retroviral vectors is their ability to integrate into the host genome, resulting in sustained transgene expression. However, with the ability to integrate comes a risk of insertional mutagenesis. Viral integration mapping studies provide an important first step in understanding the biology and fate of the vector DNA in host cells and are directly applicable to gene transfer using retroviral vectors. Using a sensitive nested PCR-based genome walking technique, we analyzed feline immunodeficiency virus (FIV)-based lentiviral vector integration events in vitro in FIV-transduced human HepG2 hepatoma cells and in vivo in the liver of mice receiving systemic injection of FIV vector. For in vitro mapping studies, we analyzed 869 sequences, which represented 211 distinct FIV integration sites. We also compared the FIV integration preferences with published results for HIV and MLV vectors. We found that ~75% of FIV integration occurred in a RefSeq gene and intron regions were preferred over exons. Of the 159 RefSeq genes with FIV integration, 139 were represented on a HepG2 microarray data set (Stanford University). The median expression level for these 139 genes was 138 and was about 3.8-fold higher than that of all the genes on the array (median expression levels for all genes on the array was 36). These data indicate that like HIV and MLV, FIV integration favors actively transcribed genes. Furthermore, similar to HIV but distinct from MLV, FIV integrated throughout the entire length of the transcriptional units, and regions around the transcription start sites were not the favored target for FIV integration. Although no specific sequences in the host genome were identified as FIV integration sites, FIV integration sites appear to be surrounded by A/T rich regions. Interestingly, FIV integrations did not coincide with the hot spots described previously for HIV integration in a human T cell line. For in vivo mapping studies, we analyzed FIV integration events in the liver of FIV-injected mice at day 5 and day 21 post gene transfer. To date, ~30 clones have been sequenced and 8 unique FIV integration sites have been identified. Five of them occurred in a gene (4 in introns and 1 in exon). The remaining 3 FIV integrations were in non-coding regions far away from transcriptional start sites. Similar mapping studies in additional animals are currently ongoing. These data suggest that although FIV integration preference is more similar to HIV than to MLV, certain differences may exist between FIV and HIV vectors. In addition, results may vary depending on the target cell type and the proliferation status of the transduced cells. These findings provide important background for understanding the host-vector interactions and stress the need for developing more site-restricted or insulated lentiviral vectors for gene transfer applications.