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Alpha-1 antitrypsin (AAT) has protective functions in animal islet transplantation models. While the therapeutic effect of AAT therapy is currently being tested in clinical trials, we investigated the mechanism of AAT protection in a clinically relevant marginal intrahepatic human islet transplantation model. In recipients receiving islets and AAT, 68.9% (20/29) reached normoglycemia, compared to 35.7% (10/28) in those receiving islets only, at 60 days posttransplant (PT). AAT-treated mice had lower serum levels of inflammatory cytokines immediately PT. Reduced M1 macrophages were observed in livers of AAT-treated recipients compared to controls as evidenced by flow cytometry and RNA-seq transcriptional profiling analysis. In vitro AAT suppressed IFN-γ-induced M1 macrophage activation/polarization via suppression of STAT1 phosphorylation and iNOS production. AAT inhibits macrophage activation induced by cytokines or dying islets, and consequently leads to islet cell survival. In a macrophage depletion mouse model, the presence of M1 macrophages in the liver contributed to graft death. AAT, through suppressing macrophage activation, protected transplanted islets from death and dysfunction in the human islet and NOD-SCID mouse model. The protective effect of AAT was confirmed in a major mismatch allogeneic islet transplantation model. Taken together, AAT suppresses liver macrophage activation that contributes to graft survival after transplantation.

Islet/b cell transplantation offers great hope for patients with type 1 diabetes. We assessed the mechanisms of how intrahepatic co-infusion of human alpha-1 antitrypsin (hAAT)-engineered mesenchymal stromal cells (hAAT-MSCs) improve survival of human islet grafts post-transplantation (PT). Longitudinal in vivo bioluminescence imaging studies identified significantly more islets in the livers bearing islets co-transplanted with hAAT-MSCs compared to islets transplanted alone. In vitro mechanistic studies revealed that hAAT-MSCs inhibit macrophage migration and suppress IFN-g-induced M1-like macrophages while promoting IL-4-induced M2-like macrophages. In vivo, this translated to significantly reduced CD11c+ and F4/80+ cells and increased CD206+ cells around islets co-transplanted with hAAT-MSCs as identified by multiplex immunofluorescence staining. Recipient-derived F4/80+and CD11b+ macrophages were mainly present in the periphery of an islet, while CD11c+ and CD206+ cells appeared inside an islet. hAAT-MSCs inhibited macrophage migration and skewed the M1-like phenotype toward an M2 phenotype both in vitro and in vivo, which may have favored islet survival. These data provide evidence that hAAT-MSCs co-transplanted with islets remain in the liver and shift macrophages to a protective state that favors islet survival. This novel strategy may be used to enhance b cell survival during islet/b cell transplantation for the treatment of type 1 diabetes or other diseases.

Islet survival is improved when islets are co-transplanted with mesenchymal stromal cells (MSCs) under the kidney capsule. We assessed whether co-infusion of MSCs with human islets into the liver, as performed in patients, would protect those islets from death post-transplantation (PT) in streptozotocin-induced diabetic NOD-SCID mice (blood glucose, BG >350mg/dl). Blood glucose levels were measured as surrogate markers for islet survival and function. Transplantation of a suboptimal islet number led to normoglycemia (BG Disclosure W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536)

We have shown that overexpression of human alpha-1 antitrypsin (hAAT) in mesenchymal stromal cells (hAAT-MSCs) improves mobility and function of MSCs. In this study we further characterized hAAT MSCs and control MSCs and compared their therapeutic effects in the prevention of type 1 diabetes in the spontaneous non-obese diabetic (NOD) mice. Bone marrow specimens from three individual donors were purchased from StemExpress. Cells were grown to passage 3-4 and infected with hAAT-containing virus for hAAT-overexpression. Single Cell RNAseq was performed by the 10X Genomic System. Proteomic Analysis of exosomes was performed at the MUSC Proteomic Core. MSCs and hAAT-MSCs from one donor were injected into the NOD mice at 8 weeks of age. Mice blood glucose levels were measured twice per week. Cell infiltration into islets was analyzed by H&E staining of pancreatic tissues. Single cell RNAseq analysis showed that hAAT-MSCs and control MSCs have different gene expression signatures, in which hAAT-MSCs showed increased expressions of chemokine (C-X-C motif) ligands (CXCL1, 3, 8), HES5 and others. Exosome proteomic analysis indicates that exosomes from these two types of cells have distinct protein patterns. In hAAT-MSCs cells, platelet degranulation proteins were upregulated and proteins involved in cell-cell adhesion, and cell division were been downregulated. In the NOD mice, a single dose of hAAT-MSCs led to delayed onset of T1D, associated with reduced cell infiltration into the islets. hAAT-MSC therapy showed more profound protection compared to control MSCs. It is likely that overexpression of hAAT enhanced the anti-inflammatory function (via regulating chemokines) promoting the Notch signaling pathway (via regulating HES5) and lead to improved protection in the NOD mice. Disclosure H. Wei: None. W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536)

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Macrophage activation contributes to islet graft loss after transplantation by secretion of proinflammatory cytokines. We showed in previous studies that treatment with alpha-1 antitrypsin (AAT) improved human islet survival after transplantation into the livers of streptozotocin-induced diabetic NOD-SCID mice. To further understand the cellular targets of AAT, we isolated macrophages from control or AAT-treated recipients at day 1 post transplantation (PT) and compared their mRNA profile using RNAseq analysis. 344 genes were changed in AAT-treated recipients compared to controls. Clusters of the inflammatory response, chemokine activity and cytokine and cytokine receptor Gene Otology categories and pathways were significantly affected by AAT-treatment. In vitro, AAT suppressed IFN-γ-induced Raw267.4 cell activation via suppression of STAT1 phosphorylation and iNOS production. This was translated in vitro when human islets were co-transplanted with IFN-γ-treated Raw cells, in the presence or absence of AAT, into diabetic NOD-SCID mice in which macrophages were depleted by liposome. At day 21, all macrophage-depleted diabetic recipients receiving islets alone achieved normoglycemia (n=6). In contrast, only 55.6% (n=9, p=0.001) receiving islets and IFN-γ-treated Raw cells achieved normoglycemia, compared to 90% receiving AAT-treated IFN-γ Raw cells (n=10, p=0.003), suggesting AAT improved islet graft survival via suppressing macrophage activation. The effect of AAT was further confirmed in a major mismatch allogeneic islet transplantation model (Balb/c, H-2b to C57BL/6 H-2d mice) in which macrophage activation contributed to primary nonfunction (PNF) of the transplanted islets. In this model, PNF occurred in 16.7% of AAT-treated recipients (n=6) but in 40% of controls (n=5) when 400 islets were transplanted. In summary, this study further identified that AAT contributed to islet graft survival after transplantation by suppressing macrophage activation. Disclosure W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (DK105183, DK120394, DK118529)

Cytokine-induced pancreatic β cell death plays a pivotal role in both type 1 and type 2 diabetes. Our previous study showed that alpha-1 antitrypsin (AAT) inhibits β cell death through the suppression of cytokine-induced c-Jun N-terminal kinase (JNK) activation in an islet transplantation model. The aim of this study was to further understand how AAT impacts β cells by studying AAT endocytosis in human islets and a βTC3 murine insulinoma cell line. Methods: In vitro, human islets and βTC3 cells were stimulated with cytokines in the presence or absence of chlorpromazine (CPZ), a drug that disrupts clathrin-mediated endocytosis. Western blot, real-time PCR and cell death ELISA were performed to investigate β cell death. The oxygen consumption rate (OCR) was measured on human islets. In vivo, islets were harvested from C57BL/6 donor mice treated with saline or human AAT and transplanted into the livers of syngeneic mice that had been rendered diabetic by streptozotocin (STZ). Islet graft survival and function were analyzed. Results: AAT was internalized by β cells in a time- and dose-dependent manner. AAT internalization was mediated by clathrin as treatment with CPZ, profoundly decreased AAT internalization, cytokine-induced JNK activation and the downstream upregulation of c-Jun mRNA expression. Similarly, addition of CPZ attenuated cytokine-induced caspase 9 cleavage (c-casp 9) and DNA fragmentation, which was suppressed by AAT. Treatment of donor mice with AAT produced AAT internalization in islets, and resulted in a higher percentage of recipients reaching normoglycemia after syngeneic intraportal islet transplantation. Conclusion: Our results suggest that AAT is internalized by β cells through clathrin-mediated endocytosis that leads to the suppression of caspase 9 activation. This process is required for the protective function of AAT in islets when challenged with proinflammatory cytokines or after islet transplantation.

Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.

Aim: Carbon monoxide (CO) functions as a therapeutic molecule in various disease models because of its anti-inflammatory and antiapoptotic properties. We investigated the capacity of CO to reduce hypoxia-induced islet cell death and dysfunction in human and mouse models. Results: Culturing islets in CO-saturated medium protected them from hypoxia-induced apoptosis and preserved β cell function by suppressing expression of proapoptotic (Bim, PARP, Cas-3), proinflammatory (TNF-α), and endoplasmic reticulum (ER) stress (glucose-regulated protein 94, grp94, CHOP) proteins. The prosurvival effects of CO on islets were attenuated when autophagy was blocked by specific inhibitors or when either ATG7 or ATG16L1, two essential factors for autophagy, was downregulated by siRNA. In vivo, CO exposure reduced both inflammation and cell death in grafts immediately after transplantation, and enhanced long-term graft survival of CO-treated human and mouse islet grafts in streptozotocin-induced diabetic non-obese diabetic severe combined immunodeficiency (NOD-SCID) or C57BL/6 recipients. Innovation: These findings underline that pretreatment with CO protects islets from hypoxia and stress-induced cell death via upregulation of ATG16L1-mediated autophagy. Conclusion: Our results suggested that CO exposure may provide an effective means to enhance survival of grafts in clinical islet cell transplantation, and may be beneficial in other diseases in which inflammation and cell death pose impediments to achieving optimal therapeutic effects. Antioxid. Redox Signal. 28, 1309–1322.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an unlimited cell source to treat cardiovascular diseases, the leading cause of death worldwide. However, current hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (∼0.5k, ∼1k, ∼3k, ∼7k cells/spheroid). Spheroids with ∼3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. Statement of Significance Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

Stresses encountered during human islet isolation lead to unavoidable β-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, β-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less β-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.

Tibetan chicken, an indigenous breed, is highly adapted to the extreme environment of the Qinghai-Tibet Plateau. It serves as a model organism to identify genetic differences between hypoxia-adapted and lowland breeds. However, the mechanisms underlying hypoxia adaptation are yet unclear. This study aimed to identify differently abundant proteins (DAPs) and elucidate the mechanisms involved in hypoxic adaptation in the Tibetan chicken. In this study, we obtained proteome data for the embryonic heart tissues of Tibetan and Chahua chickens incubated under hypoxia (TCH and CHH) and normoxia (TCN and CHN) using isobaric tags for relative and absolute quantitation (iTRAQ) technology. We identified 4210 proteins from 53,352 unique peptides in the heart tissue of chicken embryos. Pairwise TCH vs. CHH, TCH vs. TCN, CHH vs. CHN, and TCN vs. CHN comparisons revealed 118, 176, 103, and 162 differently abundant proteins, respectively. Several key proteins (EGLN1, MAP2K2, MYLK, QARS, NOTCH2, and MYH7) and pathways (glutathione metabolism, PPAR signaling pathway, and vascular smooth muscle contraction) were identified and considered important candidates for high-altitude adaptation in Tibetan chicken. This study provides novel insights into the chicken embryonic heart tissue and furthers the current understanding of the mechanisms of survival among animals in high-altitude environments.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Infiltrating macrophages can induce inflammation by secretion of proinflammatory cytokines and contribute to islet graft loss shortly after islet transplantation. In this study, we tested the mechanistic effects of AAT in mitigating macrophage mediated inflammation in an intrahepatic islet transplantation model in which human islets (2500 islet equivalent numbers) were transplanted into NOD-SCID mice that had been rendered diabetic (blood glucose >350mg/dl) by streptozotocin treatment. AAT (Prolastin-C, Grifols) was given to recipients intraperitoneally at 4 mg/mouse, every two days for 14 days, beginning before transplantation. In those recipients receiving AAT (n=29), 69.0% reached normoglycemia, compared to 35.7% in controls (n=28, p=0.002, logrank test) at 30 days post transplantation. Compared to mice treated with vehicle, AAT-treated mice had lower serum levels of interleukin 1 β (IL-1β), interferon- γ (IFN-γ) and interleukin 6 (IL-6), at day 1 post transplantation. To explore potential cell target(s) of AAT action, we analyzed activation of macrophages infiltrated into the liver after transplantation. Fewer M1 macrophages were observed in AAT-treated mice than in controls. To measure the effects of AAT on macrophage activation, we added AAT to Raw264.7 cells stimulated with IFN-γ in vitro. Expressions of M1 markers (iNOS, CD11c, TNFα, and IL-6) were dramatically decreased in AAT-treated cells compared to vehicle-treated controls. Co-culture of IFN-γ activated macrophages with islets led to islet cell apoptosis. Addition of AAT in the co-culture system reduced macrophage activation and protected islet cells from apoptosis. In summary, AAT treatment significantly enhances human islet graft survival and function after transplantation into the liver of NOD-SCID mice, in part by suppression of macrophage activation and pro-inflammatory cytokine production. Disclosure W. Gou: None. J. Wang: None. D. Kim: None. L. Song: None. C. Strange: Research Support; Self; Grifols, CSL Behring, Shire. Consultant; Self; Shire. Stock/Shareholder; Self; Abeona. Consultant; Self; CSL Behring. Research Support; Self; Adverum. Consultant; Self; Adverum. Research Support; Self; MatRx. D. Adams: None. K. Morgan: None. H. Wang: None.

The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 105 or 1 × 106 GFP+ ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP+ ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP+ ASCs migrated to pancreas and differentiated into amylase+ cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase+ cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis.

Tibetan chickens have unique adaptations to the extreme high-altitude environment that they inhabit. Epigenetic DNA methylation affects many biological processes, including hypoxic adaptation; however, the regulatory genes for DNA methylation in hypoxic adaptation remain unknown. In this study, methylated DNA immunoprecipitation with high-throughput sequencing (MeDIP-seq) was used to provide an atlas of the DNA methylomes of the heart tissue of hypoxic highland Tibetan and lowland Chahua chicken embryos. A total of 31.2 gigabases of sequence data were generated from six MeDIP-seq libraries. We identified 1,049 differentially methylated regions (DMRs) and 695 related differentially methylated genes (DMGs) between the two chicken breeds. The DMGs are involved in vascular smooth muscle contraction, VEGF signaling pathway, calcium signaling pathway, and other hypoxia-related pathways. Five candidate genes that had low methylation (EDNRA, EDNRB2, BMPR1B, BMPRII, and ITGA2) might play key regulatory roles in the adaptation to hypoxia in Tibetan chicken embryos. Our study provides significant explanations for the functions of genes and their epigenetic regulation for hypoxic adaptation in Tibetan chickens.

Tibetan chicken, unlike their lowland counterparts, exhibit specific adaptations to high-altitude conditions. The genetic mechanisms of such adaptations in highland chickens were determined by resequencing the genomes of four highland (Tibetan and Lhasa White) and four lowland (White Leghorn, Lindian, and Chahua) chicken populations. Our results showed an evident genetic admixture in Tibetan chickens, suggesting a history of introgression from lowland gene pools. Genes showing positive selection in highland populations were related to cardiovascular and respiratory system development, DNA repair, response to radiation, inflammation, and immune responses, indicating a strong adaptation to oxygen scarcity and high-intensity solar radiation. The distribution of allele frequencies of nonsynonymous single nucleotide polymorphisms between highland and lowland populations was analyzed using chi-square test, which showed that several differentially distributed genes with missense mutations were enriched in several functional categories, especially in blood vessel development and adaptations to hypoxia and intense radiation. RNA sequencing revealed that several differentially expressed genes were enriched in gene ontology terms related to blood vessel and respiratory system development. Several candidate genes involved in the development of cardiorespiratory system (FGFR1, CTGF, ADAM9, JPH2, SATB1, BMP4, LOX, LPR, ANGPTL4, and HYAL1), inflammation and immune responses (AIRE, MYO1F, ZAP70, DDX60, CCL19, CD47, JSC, and FAS), DNA repair, and responses to radiation (VCP, ASH2L, and FANCG) were identified to play key roles in the adaptation to high-altitude conditions. Our data provide new insights into the unique adaptations of highland animals to extreme environments.

Heme oxygenase 1 (HO-1), a rate-limiting enzyme of heme catabolism, has a crucial role of cytoprotective functions under hypoxia. The objective of the present study was to investigate potential differences in protective effect of HO-1 gene on chicken (Gallus gallus) embryo lung during late incubation. At embryonic day (D) D16, D18, D19, and D20 of incubation, the expression of HO-1 in the lungs of chicken embryos (Tibet and Shouguang chickens) incubated in normoxic (21% O2) and hypoxic (13% O2) conditions was measured. SNPs were screened within 5′-flanking region and coding regions with PCR-sequencing and the genotype of the SNPs was determined with PCR-RFLP in Tibet, Chahua and Shouguang chicken populations. In conclusion, the Tibet chicken had higher HO-1 expression on D19 under hypoxic incubation and had two SNPs with different frequency distributions from other chicken breeds, which might be a way that the Tibet chicken had hereditary adaptation to hypoxia during embryonic development.

Nitric oxide (NO), an endothelium-derived relaxing factor, is synthesized from l -arginine by endothelial nitric oxide synthase (eNOS) in the endothelium. The objective of the present study was to preliminarily illuminate the expression of the eNOS gene in hypoxic adaptation of chicken embryonic development. The eNOS expression profiles between the Tibet and Shouguang chickens incubated under both normoxic and hypoxic conditions were detected by TaqMan real-time PCR. In this study, the chicken eNOS gene was found by both in silico cloning and RACE approaches. From the eNOS gene, we obtained a 3,310-bp mRNA sequence and a 10,666-bp DNA sequence and discovered that it was located on chicken chromosome 2 and had 7 unique transcripts. eNOS mRNA was detected in abundant amounts in some chick embryo organs (i.e., heart, liver, chorio-allantoic membrane, and lung), and expressed stably with the lowest levels in the brain. We observed that when exposed to hypoxia (13% O2) different embryo organ tissues had various sensitivities to hypoxia as determined by their eNOS expression profiles. Compared with the Shouguang chicken, the eNOS expression in the Tibet chicken was higher in the lung and liver, lower in the heart, and similar in the brain. In chorio-allantoic membranes, eNOS expression was higher in the Shouguang chicken than the Tibet chicken under hypoxic conditions, but not markedly different under normoxic conditions. The differences of eNOS expression between the 2 breeds may be relative to the hypoxic adaptation ability in Tibet chickens during embryonic development. This work will provide reference for future studies on the role of eNOS in hypoxic adaptation and response.