publications

2024

1. Integrative gene regulatory network analysis discloses key driver genes of fibromuscular dysplasia

   Valentina d’Escamard, Daniella Kadian-Dodov, Lijiang Ma, and 30 more authors

   Nat Cardiovasc Res, Sep 2024

   Publisher: Nature Publishing Group

   Abs DOI

   Fibromuscular dysplasia (FMD) is a poorly understood disease affecting 3–5% of adult females. The pathobiology of FMD involves arterial lesions of stenosis, dissection, tortuosity, dilation and aneurysm, which can lead to hypertension, stroke, myocardial infarction and even death. Currently, there are no animal models for FMD and few insights as to its pathobiology. In this study, by integrating DNA genotype and RNA sequence data from primary fibroblasts of 83 patients with FMD and 71 matched healthy controls, we inferred 18 gene regulatory co-expression networks, four of which were found to act together as an FMD-associated supernetwork in the arterial wall. After in vivo perturbation of this co-expression supernetwork by selective knockout of a top network key driver, mice developed arterial dilation, a hallmark of FMD. Molecular studies indicated that this supernetwork governs multiple aspects of vascular cell physiology and functionality, including collagen/matrix production. These studies illuminate the complex causal mechanisms of FMD and suggest a potential therapeutic avenue for this challenging disease.

2023

1. The TNFΔARE Mouse as a Model of Intestinal Fibrosis

   Calen A. Steiner, Samuel D. Koch, Tamara Evanoff, and 11 more authors

   The American Journal of Pathology, Aug 2023

   Abs DOI

   Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease–related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At \textgreater24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

2. Novel EGFR-mutant mouse models of lung adenocarcinoma reveal adaptive immunity requirement for durable osimertinib response

   Emily K. Kleczko, Anh T. Le, Trista K. Hinz, and 10 more authors

   Cancer Letters, Mar 2023

   Abs DOI

   Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer patient biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu or Rag1−/− mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral T cell content and decreased neutrophil content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

3. Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis

   Austin J. Jolly, Sizhao Lu, Allison M. Dubner, and 8 more authors

   JCI Insight, Mar 2023

   Publisher: American Society for Clinical Investigation

   Abs DOI

   Vascular smooth muscle–derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM–derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM–to–myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1–induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM–to–myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

4. Smooth muscle–derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner

   Allison M. Dubner, Sizhao Lu, Austin J. Jolly, and 8 more authors

   JCI Insight, Nov 2023

   Publisher: American Society for Clinical Investigation

   Abs DOI

   We previously established that vascular smooth muscle–derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM–derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non–AdvSca1-SM–derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.

5. Confounding Effects of Tamoxifen: Cautionary and Practical Considerations for the Use of Tamoxifen-Inducible Mouse Models in Atherosclerosis Research—Brief Report

   Allison M. Dubner, Sizhao Lu, Austin J. Jolly, and 6 more authors

   Arteriosclerosis, Thrombosis, and Vascular Biology, Nov 2023

   Publisher: American Heart Association

   Abs DOI

   BACKGROUND: In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most frequently used lineage tracing approaches involves tamoxifen-inducible CreERT-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored. METHODS: To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured. RESULTS: After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil–treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation. CONCLUSIONS: In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.

6. IL-6 mediates the hepatic acute phase response after prerenal azotemia in a clinically defined murine model

   Kayo Okamura, Sizhao Lu, Zhibin He, and 7 more authors

   American Journal of Physiology-Renal Physiology, Sep 2023

   Publisher: American Physiological Society

   Abs DOI

   Prerenal azotemia (PRA) is a major cause of acute kidney injury and uncommonly studied in preclinical models. We sought to develop and characterize a novel model of PRA that meets the clinical definition: acute loss of glomerular filtration rate (GFR) that returns to baseline with resuscitation. Adult male C57BL/6J wild-type (WT) and IL-6−/− mice were studied. Intraperitoneal furosemide (4 mg) or vehicle was administered at time = 0 and 3 h to induce PRA from volume loss. Resuscitation began at 6 h with 1 mL intraperitoneal saline for four times for 36 h. Six hours after furosemide administration, measured glomerular filtration rate was 25% of baseline and returned to baseline after saline resuscitation at 48 h. After 6 h of PRA, plasma interleukin (IL)-6 was significantly increased, kidney and liver histology were normal, kidney and liver lactate were normal, and kidney injury molecule-1 immunofluorescence was negative. There were 327 differentially regulated genes upregulated in the liver, and the acute phase response was the most significantly upregulated pathway; 84 of the upregulated genes (25%) were suppressed in IL-6−/− mice, and the acute phase response was the most significantly suppressed pathway. Significantly upregulated genes and their proteins were also investigated and included serum amyloid A2, serum amyloid A1, lipocalin 2, chemokine (C-X-C motif) ligand 1, and haptoglobin; hepatic gene expression and plasma protein levels were all increased in wild-type PRA and were all reduced in IL-6−/− PRA. This work demonstrates previously unknown systemic effects of PRA that includes IL-6-mediated upregulation of the hepatic acute phase response.

2022

1. Lgals3-Transitioned Inflammatory Smooth Muscle Cells: Major Regulators of Atherosclerosis Progression and Inflammatory Cell Recruitment

   Sizhao Lu, and Mary C. M. Weiser-Evans

   Arterioscler Thromb Vasc Biol, Aug 2022

   DOI
2. Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodelling

   Austin J Jolly, Sizhao Lu, Keith A Strand, and 6 more authors

   Cardiovascular Research, May 2022

   Abs DOI

   Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodelling events, such as neointima formation, atherosclerotic lesion development, and adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signalling mechanisms that underlie remodelling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodelling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodelling cells, such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodelling. Important questions remain regarding the origins of AdvSca1 cells and the essential signalling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations, which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodelling, and comment on their translational relevance in humans.

2020

1. High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog)

   Keith A. Strand, Sizhao Lu, Marie F. Mutryn, and 10 more authors

   Arteriosclerosis, Thrombosis, and Vascular Biology, Aug 2020

   Publisher: American Heart Association

   Abs DOI

   Download figureDownload PowerPoint

2. Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis

   Sizhao Lu, Austin J. Jolly, Keith A. Strand, and 6 more authors

   JCI Insight, Dec 2020

   Publisher: American Society for Clinical Investigation

   Abs DOI

   Resident vascular adventitial SCA1+ progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage-tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA-sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling were significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM-specific expression of GLI-Kruppel family member GLI1 (Gli1), we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell-specific Kruppel-like factor 4 (Klf4) depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a profibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach.

3. PTEN (Phosphatase and Tensin Homolog) Protects Against Ang II (Angiotensin II)-Induced Pathological Vascular Fibrosis and Remodeling-Brief Report

   Sizhao Lu, Keith A. Strand, Marie F. Mutryn, and 6 more authors

   Arterioscler Thromb Vasc Biol, Feb 2020

   Abs DOI

   OBJECTIVE: Pathological vascular remodeling and excessive perivascular fibrosis are major contributors to reduced vessel compliance that exacerbates cardiovascular diseases, for instance, promoting clinically relevant myocardial remodeling. Inflammation plays a significant role in both pathological vascular remodeling and fibrosis. We previously demonstrated that smooth muscle cell-specific PTEN depletion promotes significant vascular fibrosis and accumulation of inflammatory cells. In the current study, we aimed to determine the beneficial role of systemic PTEN elevation on Ang II (angiotensin II)-induced vascular fibrosis and remodeling. Approach and Results: Transgenic mice carrying additional copies of the wild-type Pten gene (super PTEN [sPTEN]) and WT littermates were subjected to Ang II or saline infusion for 14 or 28 days. Compared with WT, Ang II-induced vascular fibrosis was significantly blunted in sPTEN mice, as shown by histochemical stainings and label-free second harmonic generation imaging. The protection against Ang II was recapitulated in sPTEN mice bearing WT bone marrow but not in WT mice reconstituted with sPTEN bone marrow. Ang II-induced elevation of profibrotic and proinflammatory gene expression observed in WT mice was blocked in aortic tissue of sPTEN mice. Immunofluorescent staining and flow cytometry both indicated that perivascular infiltration of T cells and macrophages was significantly inhibited in sPTEN mice. In vitro induction of PTEN expression suppressed Ang II-induced Ccl2 expression in vascular smooth muscle cells. CONCLUSIONS: Systemic PTEN elevation mediates protection against Ang II-induced vascular inflammation and fibrosis predominantly through effects in resident vascular cells. Our data highly support that pharmacological upregulation of PTEN could be a novel and viable approach for the treatment of pathological vascular fibrosis.

4. Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype

   James F. Dylewski, Nancy Wilson, Sizhao Lu, and 4 more authors

   MethodsX, Jan 2020

   Abs DOI

   Glomerular endothelial cells (GEnC) are a specialized microvascular subset of endothelial cells that, when injured, result in many types of diseases within the kidney. Thus, techniques to study GEnC in a cell culture system are important to investigate mechanisms of GEnC injury. Studies of endothelial cell function in culture have predominately relied on using macrovascular endothelial cells from vascular areas other than the glomerulus. Over the last 15 years, glomerular endothelial cells lines have been created but were isolated by targeting cells expressing CD31. Some studies identified endothelial cells isolated from the microvasculature do not express CD31 and some suggest that CD31+ cells are phenotypically different than endothelial cells found in capillaries. Here we detail our method of isolation, purification, and conditional immortalization of mouse glomerular endothelial cells targeting endothelial cells that do not express CD31.•This method allows for isolation, purification, and conditional immortalization of glomerular endothelial cells for continued passage of GEnCs beyond that of primary cell culture.•This method can be used in genetically modified mice to investigate how a modification of a specific gene or protein affects the glomerular endothelium at the cellular level.

2019

1. Nuclear Focal Adhesion Kinase

   Lu Sizhao, and  Weiser-Evans Mary C.M.

   Circulation Research, Jul 2019

   DOI

2018

1. PTEN deficiency promotes pathological vascular remodeling of human coronary arteries

   Karen S. Moulton, Marcella Li, Keith Strand, and 10 more authors

   JCI Insight, Feb 2018

   Abs DOI

   Phosphatase and tensin homolog (PTEN) is an essential regulator of the differentiated vascular smooth muscle cell (SMC) phenotype. Our goal was to establish that PTEN loss promotes SMC dedifferentiation and pathological vascular remodeling in human atherosclerotic coronary arteries and nonatherosclerotic coronary arteries exposed to continuous-flow left ventricular assist devices (CF-LVADs). Arteries were categorized as nonatherosclerotic hyperplasia (NAH), atherosclerotic hyperplasia (AH), or complex plaque (CP). NAH coronary arteries from CF-LVAD patients were compared to NAH coronaries from non-LVAD patients. Intimal PTEN and SMC contractile protein expression was reduced compared with the media in arteries with NAH, AH, or CP. Compared with NAH, PTEN and SMC contractile protein expression was reduced in the media and intima of arteries with AH and CP. NAH arteries from CF-LVAD patients showed marked vascular remodeling and reduced PTEN and α-smooth muscle actin (αSMA) in medial SMCs compared with arteries from non-LVAD patients; this correlated with increased medial collagen deposition. Mechanistically, compared with ApoE–/– mice, SMC-specific PTEN-null/ApoE–/– double-knockout mice exhibited accelerated atherosclerosis progression and increased vascular fibrosis. By microarray and validated quantitative RT-PCR analysis, SMC PTEN deficiency promotes a global upregulation of proinflammatory and profibrotic genes. We propose that PTEN is an antiinflammatory, antifibrotic target that functions to maintain SMC differentiation. SMC loss of PTEN results in pathological vascular remodeling of human arteries.

2017

1. FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis

   Sathish Kumar Natarajan, Bailey A. Stringham, Ashley M. Mohr, and 5 more authors

   J. Lipid Res., May 2017

   Abs DOI

   Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

2. Differentiated Smooth Muscle Cells Generate a Subpopulation of Resident Vascular Progenitor Cells in the Adventitia Regulated by Klf4

   Mark W. Majesky, Henrick Horita, Allison Ostriker, and 7 more authors

   Circulation Research, Jan 2017

   Abs DOI

   Rationale:The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (adventitial sca1-positive progenitor cells [AdvSca1]), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo.Objective:Although emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and heterogeneity, and contribution to vessel remodeling.Methods and Results:Using 2 in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. After vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo, whereas in vitro approaches demonstrate that Klf4 is essential for the maintenance of the AdvSca1 progenitor phenotype.Conclusions:We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

2016

1. Lack of hepcidin expression attenuates steatosis and causes fibrosis in the liver

   Sizhao Lu, Robert G. Bennett, Kusum K. Kharbanda, and 1 more author

   World J Hepatol, Feb 2016

   Abs DOI

   AIM: To investigate the role of key iron-regulatory protein, hepcidin in non-alcoholic fatty liver disease (NAFLD). METHODS: Hepcidin (Hamp1) knockout and floxed control mice were administered a high fat and high sucrose (HFS) or a regular control diet for 3 or 7 mo. Steatosis, triglycerides, fibrosis, protein and gene expression in mice livers were determined by histological and biochemical techniques, western blotting and real-time polymerase chain reaction. RESULTS: Knockout mice exhibited hepatic iron accumulation. Despite similar weight gains, HFS feeding induced hepatomegaly in floxed, but not knockout, mice. The livers of floxed mice exhibited higher levels of steatosis, triglycerides and c-Jun N-terminal kinase (JNK) phosphorylation than knockout mice. In contrast, a significant increase in fibrosis was observed in knockout mice livers within 3 mo of HFS administration. The hepatic gene expression levels of sterol regulatory element-binding protein-1c and fat-specific protein-27, but not peroxisome proliferator-activated receptor-alpha or microsomal triglyceride transfer protein, were attenuated in HFS-fed knockout mice. Knockout mice fed with regular diet displayed increased carnitine palmitoyltransferase-1a and phosphoenolpyruvate carboxykinase-1 but decreased glucose-6-phosphatase expression in the liver. In summary, attenuated steatosis correlated with decreased expression of lipogenic and lipid storage genes, and JNK phosphorylation. Deletion of Hamp1 alleles per se modulated hepatic expression of beta-oxidation and gluconeogenic genes. CONCLUSION: Lack of hepcidin expression inhibits hepatic lipid accumulation and induces early development of fibrosis following high fat intake. Hepcidin and iron may play a role in the regulation of metabolic pathways in the liver, which has implications for NAFLD pathogenesis.

2. Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway

   Sizhao Lu, Sathish Kumar Natarajan, Justin L. Mott, and 2 more authors

   PLOS ONE, Jan 2016

   Publisher: Public Library of Science

   Abs DOI

   Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

2015

1. Inductively coupled mass spectrometry analysis of biometals in conditional Hamp1 and Hamp1 and Hamp2 transgenic mouse models

   S. Lu, J. Seravalli, and D. Harrison-Findik

   Transgenic Res, Apr 2015

   Abs DOI

   Hepcidin, a circulatory antimicrobial peptide, is involved in iron homeostasis, inflammation, infection and metabolic signals. Humans express one hepcidin gene, HAMP but mice express two hepcidin genes, Hamp1 and Hamp2. Consecutive gene targeting events were performed to produce transgenic mice expressing conditional alleles of either Hamp1 or both Hamp1 and Hamp2 (Hamp1/2). The deletion of Hamp1 alleles elevated Hamp2 expression, particularly in males, which was reduced by endotoxin treatment. The tissue levels of iron and other biometals were quantified by inductively coupled mass spectrometry. The ubiquitous or liver-specific deletion of Hamp1 alleles yielded similar quantitative changes in iron levels in the liver, duodenum, spleen, kidney, heart and brain. The introduction of Hamp2 null allele did not exacerbate the iron-related phenotype of Hamp1 null allele. Besides iron, Hamp1 null allele significantly elevated the levels of selenium in the liver, manganese in the liver and duodenum, and copper in the brain. Mice with conditional Hamp alleles will be useful to determine the tissue-specific regulation and functions of Hamp1 and Hamp2 in biometal homeostasis and other biological processes.

2. Saturated Fatty Acids Induce Post-transcriptional Regulation of HAMP mRNA via AU-rich Element-binding Protein, Human Antigen R (HuR)

   Sizhao Lu, Justin L. Mott, and Duygu Dee Harrison-Findik

   J. Biol. Chem., Oct 2015

   Publisher: American Society for Biochemistry and Molecular Biology

   Abs DOI

   Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3′-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3′-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3′-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

3. The Effect of Alcohol and Hydrogen Peroxide on Liver Hepcidin Gene Expression in Mice Lacking Antioxidant Enzymes, Glutathione Peroxidase-1 or Catalase

   Duygu Dee Harrison-Findik, and Sizhao Lu

   Biomolecules, Jun 2015

   Number: 2 Publisher: Multidisciplinary Digital Publishing Institute

   Abs DOI

   This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1−/−) and catalase (catalase−/−) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1−/− displayed significantly higher hepatic H2O2 levels than catalase−/− compared to wild-type mice, as measured by 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1−/− mice. Alcohol increased H2O2 production in catalase−/− and wild-type, but not gpx-1−/−, mice. Hepcidin expression was inhibited in alcohol-fed catalase−/− and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1−/− mice. Gpx-1−/− mice also displayed higher level of basal liver CHOP protein expression than catalase−/− mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1−/− mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1−/− mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

4. Transcriptional and Post-transcriptional Regulation of Hepcidin and Iron Metabolism by Lipid Signaling in the Liver

   Sizhao Lu

   Theses & Dissertations, Aug 2015

2014

1. TLR4 signaling and the inhibition of liver hepcidin expression by alcohol

   Emily Zmijewski, Sizhao Lu, and Duygu Dee Harrison-Findik

   World J Gastroenterol, Sep 2014

   Abs DOI

   AIM: To understand the role of toll-like receptor 4 (TLR4) signaling in the regulation of iron-regulatory hormone, hepcidin by chronic alcohol consumption., METHODS: For chronic alcohol intake studies, TLR4 mutant mice on C3H/HeJ background and wildtype counterpart on C3H/HeOuJ background were pair-fed with regular (control) and ethanol-containing Lieber De Carli liquids diets. Gene expression was determined by real-time quantitative PCR. Protein-protein interactions and protein expression were determined by co-immunoprecipitation and western blotting. The occupancy of hepcidin gene promoter was determined by chromatin immunoprecipitation assays., RESULTS: Chronic alcohol intake suppressed hepcidin mRNA expression in the livers of wildtype, but not TLR4 mutant, mice. The phosphorylation and nuclear translocation of nuclear factor (NF)-κB p65 subunit protein was observed in alcohol-fed wildtype, but not in alcohol-fed TLR4 mutant, mice. Similarly, alcohol induced the binding of NF-κB p50 subunit protein to hepcidin gene promoter in wildtype, but not in TLR4 mutant, mice. In contrast, the phosphorylation of Stat3 in the liver was stronger in alcohol-treated TLR4 mutant mice compared to alcohol-treated wildtype mice. The occupancy of hepcidin gene promoter by Stat3 was observed in alcohol-fed mutant, but not in wildtype, mice. An interaction between NF-κB p65 subunit protein and small heterodimer partner protein (SHP) was observed in the livers of both wildtype and TLR4 mutant mice fed with the control diet, as shown by co-immunoprecipitation studies. Alcohol intake elevated cytosolic SHP expression but attenuated its interaction with NF-κB in the liver, which was more prominent in the livers of wildtype compared to TLR4 mutant mice., CONCLUSION: Activation of TLR4 signaling and NF-кB are involved in the suppression of hepcidin gene transcription by alcohol in the presence of inflammation in the liver.

2. Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression

   Sizhao Lu, Emily Zmijewski, John Gollan, and 1 more author

   World J Biol Chem, Aug 2014

   Abs DOI

   AIM: To determine the regulation of human hepcidin (HAMP) and mouse hepcidin (hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models., METHODS: For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR (qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation (ChIP) assays., RESULTS: The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3 (P \textless 0.001), but did not cause any significant changes in HAMP expression. Short-term (1 h) Jo2 treatment (0.2 μg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis (P \textless 0.001) and an increase in SAA3 (P \textless 0.023) and IL-6 (P \textless 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment (0.2 μg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2 (0.2 μg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2 (0.32 μg/g b.w.) did not also alter hepatic hepcidin expression., CONCLUSION: Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver.

2013

1. Autophagy and cancer

   Si-Zhao Lu, and Duygu Dee Harrison-Findik

   World J Biol Chem, Aug 2013

   Abs DOI

   Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles. Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion, energy deficiency or the upregulation of Bcl-2 protein expression. A novel role of autophagy in various cancers has been proposed. Interestingly, evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported. As a tumor suppression mechanism, autophagy maintains genome stability, induces senescence and possibly autophagic cell death. On the other hand, autophagy participates in tumor growth and maintenance by supplying metabolic substrate, limiting oxidative stress, and maintaining cancer stem cell population. It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific. In addition, substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer, and represents one of the potential directions for future studies.

2. Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression

   Duygu Dee Harrison-Findik, Sizhao Lu, Emily M Zmijewski, and 2 more authors

   World J Biol Chem, Nov 2013

   Abs DOI

   AIM: To understand the role of mitochondrial-produced superoxide (O2•-) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver., METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2+/-) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w/v) ethanol in the drinking water or plain water (control) for 7 d. Total cellular O2•- levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2•–sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2•- in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively., RESULTS: Sod2+/- mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2+/- and LMC mice. O2•- levels in hepatocytes of untreated Sod2+/- mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2•- levels in mitochondria of Sod2+/ mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O2•- levels in hepatocytes of LMC mice than in Sod2+/- mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O2•- levels in both Sod2+/- and control mice., CONCLUSION: Mitochondrial O2•- is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O2•- levels in hepatocytes, which appears not to originate from mitochondria.

2012

1. Alcohol Activates TGF-Beta but Inhibits BMP Receptor-Mediated Smad Signaling and Smad4 Binding to Hepcidin Promoter in the Liver

   Lisa Nicole Gerjevic, Na Liu, Sizhao Lu, and 1 more author

   International Journal of Hepatology, Nov 2012

   _eprint: https://onlinelibrary.wiley.com/doi/pdf/10.1155/2012/459278

   Abs DOI

   Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs). Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

2011

1. Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

   Lisa Nicole Gerjevic, Sizhao Lu, Jonathan Pascal Chaky, and 1 more author

   World J Biol Chem, Dec 2011

   Abs DOI

   AIM: To study the effect of both acute and chronic alcohol exposure on heme oxygenases (HOs) in the brain, liver and duodenum., METHODS: Wild-type C57BL/6 mice, heterozygous Sod2 knockout mice, which exhibit attenuated manganese superoxide dismutase activity, and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia. For acute alcohol exposure, ethanol was administered in the drinking water for 1 wk. Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies. HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting., RESULTS: Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice. It did not alter liver HO-1 expression, nor HO-2 expression in the brain, liver or duodenum. In contrast, acute alcohol exposure decreased both liver HO-1 and HO-2 expression, and HO-2 expression in the duodenum of wild-type mice. The decrease in liver HO-1 expression was abolished in ARNT+/- mice. Sod2+/- mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression. However, alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/- mice. Collectively, these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner. Chronic alcohol exposure alters brain and duodenal, but not liver HO expression. However, acute alcohol exposure inhibits liver HO-1 and HO-2, and also duodenal HO-2 expression., CONCLUSION: The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.