Protective Role for Smooth Muscle Cell Hepcidin in Abdominal Aortic Aneurysm.

BACKGROUND: Hepcidin is a liver-derived hormone that controls systemic iron homeostasis, by inhibiting the iron exporter ferroportin in the gut and spleen, respective sites of iron absorption and recycling. Hepcidin is also expressed ectopically in the context of cardiovascular disease. However, the precise role of ectopic hepcidin in underlying pathophysiology is unknown. In patients with abdominal aortic aneurysm (AAA), hepcidin is markedly induced in smooth muscle cells (SMCs) of the aneurysm wall and inversely correlated with the expression of LCN2 (lipocalin-2), a protein implicated in AAA pathology. In addition, plasma hepcidin levels were inversely correlated with aneurysm growth, suggesting hepcidin has a potential disease-modifying role. METHODS: To probe the role of SMC-derived hepcidin in the setting of AAA, we applied AngII (Angiotensin-II)-induced AAA model to mice harbouring an inducible, SMC-specific deletion of hepcidin. To determine whether SMC-derived hepcidin acted cell-autonomously, we also used mice harboring an inducible SMC-specific knock-in of hepcidin-resistant ferroportinC326Y. The involvement of LCN2 was established using a LCN2-neutralizing antibody. RESULTS: Mice with SMC-specific deletion of hepcidin or knock-in of hepcidin-resistant ferroportinC326Y had a heightened AAA phenotype compared with controls. In both models, SMCs exhibited raised ferroportin expression and reduced iron retention, accompanied by failure to suppress LCN2, impaired autophagy in SMCs, and greater aortic neutrophil infiltration. Pretreatment with LCN2-neutralizing antibody restored autophagy, reduced neutrophil infiltration, and prevented the heightened AAA phenotype. Finally, plasma hepcidin levels were consistently lower in mice with SMC-specific deletion of hepcidin than in controls, indicating that SMC-derived hepcidin contributes to the circulating pool in AAA. CONCLUSIONS: Hepcidin elevation in SMCs plays a protective role in the setting of AAA. These findings are the first demonstration of a protective rather than deleterious role for hepcidin in cardiovascular disease. They highlight the need to further explore the prognostic and therapeutic value of hepcidin outside disorders of iron homeostasis.

Fetal liver hepcidin secures iron stores in utero.

In the adult, the liver-derived hormone hepcidin (HAMP) controls systemic iron levels by blocking the iron-exporting protein ferroportin (FPN) in the gut and spleen, the sites of iron absorption and recycling, respectively. Impaired HAMP expression or FPN responsiveness to HAMP result in iron overload. HAMP is also expressed in the fetal liver but its role in controlling fetal iron stores is not understood. To address this question in a manner that safeguards against the confounding effects of altered maternal iron homeostasis, we generated fetuses harboring a paternally-inherited ubiquitous knock-in of the HAMP-resistant fpnC326Y. Additionally, to safeguard against any confounding effects of altered placental iron homeostasis, we generated fetuses with a liver-specific knock-in of fpnC326Y or knockout of the hamp gene. These fetuses had reduced liver iron stores and hemoglobin, and markedly increased FPN in the liver, but not in the placenta. Thus, fetal liver HAMP operates cell-autonomously to increase fetal liver iron stores. Our findings also suggest that FPN in the placenta is not actively regulated by fetal liver HAMP under normal physiological conditions.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

The kidney hepcidin/ferroportin axis controls iron reabsorption and determines the magnitude of kidney and systemic iron overload.

The hepcidin/ferroportin axis controls systemic iron homeostasis by regulating iron acquisition from the duodenum and reticuloendothelial system, respective sites of iron absorption and recycling. Ferroportin is also abundant in the kidney, where it has been implicated in tubular iron reabsorption. However, it remains unknown whether endogenous hepcidin regulates ferroportin-mediated iron reabsorption under physiological conditions, and whether such regulation is important for kidney and/or systemic iron homeostasis. To address these questions, we generated a novel mouse model with an inducible kidney-tubule specific knock-in of fpnC326Y, which encodes a hepcidin-resistant ferroportin termed FPNC326Y. Under conditions of normal iron availability, female mice harboring this allele had consistently decreased kidney iron but only transiently increased systemic iron indices. Under conditions of excess iron availability, male and female mice harboring this allele had milder kidney iron overload, but greater systemic iron overload relative to controls. Additionally, despite comparable systemic iron overload, kidney iron overload occurred in wild type mice fed an iron-loaded diet but not in hemochromatosis mice harboring a ubiquitous knock-in of fpnC326Y. Thus, our study demonstrates that endogenous hepcidin controls ferroportin-mediated tubular iron reabsorption under physiological conditions. It also shows that such control is important for both kidney and systemic iron homeostasis in the context of iron overload.

Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer.

Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.

Ex Vivo PD-L1/PD-1 Pathway Blockade Reverses Dysfunction of Circulating CEA-Specific T Cells in Pancreatic Cancer Patients.

Purpose: Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if ex vivo PD-L1 and TIM-3 blockade could enhance CTL function.Experimental Design: CD8+ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2+ patients with pancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.Results: Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.Conclusions: These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. Clin Cancer Res; 23(20); 6178-89. ©2017 AACR.

A Novel Approach to Ultrasound-Mediated Tissue Decellularization and Intra-Hepatic Cell Delivery in Rats.

Liver transplantation is the mainstay of treatment for end stage liver diseases, including metabolic and congenital liver diseases. The number of suitable donor organs is, however, limited, and a whole-liver transplant requires complex surgery. Cell therapy, such as intra-portal hepatocytes transplantation, has been considered as a bridging therapy to liver transplantation but has shown a mixed clinical outcome with limited success, including low level of engraftment of transplanted hepatocytes. Here, we report a novel cell delivery technique in a rat model by creating a cavity inside the liver parenchyma by non-invasive high intensity focused ultrasound histotripsy. Our in vivo experimental results together with histologic observations show that direct injection of cells inside the cavity can facilitate successful uptake, proliferation and integration of the transplanted hepatocytes in the recipient liver. We were able to restore the plasma albumin level to 50% of the normal level in Nagase analbuminemic rats (serum albumin level of the Nagase rats was initially nil) by cell therapy after high intensity focused ultrasound-mediated histotripsy. We believe that this novel technique would enable the delivery of a large number of cells into the liver to restore liver function, particularly as a treatment for metabolic liver diseases. This novel method of intra-hepatic hepatocyte transplantation might be an invaluable tool for cell therapy in the future.

Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome.

Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.

A novel rat model of liver regeneration: possible role of cytokine induced neutrophil chemoattractant-1 in augmented liver regeneration.

BACKGROUND: Liver resection is the mainstay of treatment for most of the liver tumors. Liver has a unique capability to restore the lost volume following resection, however, most of the primary tumors grow in a liver with preexisting parenchymal diseases and secondary tumors often present in multiple liver lobes precluding a safe curative resection. Two-stage hepatectomy and portal vein ligation (PVL) are used to achieve a safer future remnant liver volume (FRLV), however, these procedures take several weeks to achieve adequate FRLV. A recently introduced faster alternative two-stage hepatectomy, also know as associated liver partitioning and portal vein ligation for staged hepatectomy (ALPPS), produces a desirable FRLV in days. METHODS: To have an insight into the mechanism of ALPPS associated liver regeneration, we reproduced a rat model of ALPPS and compared the results with the PVL group. RESULTS: Our results convincingly showed an advantage of the ALPPS procedure over PVL group in terms of early regeneration, however, in 1-week time the amount of regeneration was comparable. An early regeneration in the ALPPS group coincided with an early entry of hepatocytes into the cell proliferation phase, a significant increase in portal pressure and increase in hepatic enzymes in the ALPPS group compared with the PVL group. According to the protein array evaluation of 29 cytokines/chemokines, cytokine induced neutrophil chemoattractant-1 had the highest expression whereas IL-6 had the highest fold (>6 vs PVL group) expression at the early phase of regeneration in the ALPPS group. CONCLUSIONS: This unique rat model of ALPPS would help to improve our understanding about the liver generation process and also will help in further refinement of the ALPPS procedure for the clinical benefit.