TGF-β Signaling in Stromal Cells Contributes to Myelofibrosis, but Not Niche Disruption, in Myeloproliferative Neoplasms

Myeloproliferative neoplasms are associated with significant alterations in the bone marrow microenvironment that contribute to disease pathogenesis. The most striking alteration is the development of myelofibrosis, which is characterized by extensive collagen deposition in the bone marrow and is associated with a poor prognosis. Recent evidence suggests that expression of key niche factors, including CXCL12 (stromal derived factor-1, SDF-1) and Kit ligand are reduced in MPNs. This is relevant, since studies by our group and others have shown that deleting these niche factors from stromal cells results in a shift in hematopoiesis from the bone marrow to spleen. Indeed, a prominent feature of MPN is the development of splenomegaly and extramedullary hematopoiesis. There is evidence implicating inflammatory mediators in the development of myelofibrosis. In particular, increased production of TGF-β produced by megakaryocytes and monocytes is found in most patients with MPNs. To assess the role of TGF-β signaling in mesenchymal stromal cells in the bone marrow in the development of myelofibrosis, we generated Osx-Cre; Tgfbr2 f/- mice, in which TGF-β signaling is abrogated in all bone marrow mesenchymal stromal cells (including Lepr + stromal cells), but not endothelial cells or hematopoietic cells. We transplanted MPL W515L transduced hematopoietic stem and progenitor cells (HSPCs) or JAK2 V617F bone marrow into these mice and quantified myelofibrosis using reticulin staining and Collagen 1 and 3 immunostaining. We previously reported that deletion of TGF-β signaling in mesenchymal stromal cells in these mice abrogated the development of myelofibrosis, and we presented evidence that this was mediated by non-canonical JNK-dependent TGF-β signaling. Here, we describe the impact of stromal TGF-β signaling on the bone marrow hematopoietic niche in MPN. MPL W515L transduced HSPCs were transplanted into Osx-Cre; Tgfbr2 f/- mice, and the impact on hematopoietic niche disruption and development of extramedullary hematopoiesis was assessed. In control recipients, transplantation of MPL W515L HSPCs resulted in marked decreases in bone marrow Cxcl12 and Kit ligand expression (Figure 1A-B). Surprisingly, a similar decrease was observed in Osx-Cre; Tgfbr2 f/- recipients. The loss of these key niche factors is predicted to impair hematopoietic niche function in the bone marrow. Consistent with this prediction, total bone marrow cellularity and HSC number were significantly reduced in both control and Osx-Cre; Tgfbr2 f/- recipients (Figure 1C-D). Finally, disruption of the bone marrow niche is often associated with extramedullary hematopoiesis. Indeed, a significant increase in spleen size and spleen HSCs and erythroid progenitors was observed in control recipients (Figure 1E-G). Again, a similar phenotype was observed in Osx-Cre; Tgfbr2 f/- recipients. Collectively, these data show that TGF-β signaling in bone marrow mesenchymal stromal cells is required for the development of myelofibrosis but not hematopoietic niche disruption in MPNs. Thus, these data show for the first time that the signals that induce a fibrogenic program in bone marrow mesenchymal stromal cells are distinct from those that suppress Cxcl12 and Kit ligand expression. Our data show that the fibrogenic program is dependent on non-canonical JNK-dependent TGF-β signaling, while the signals that regulate niche factor expression remain unknown. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract P110: Proteomics Identified Novel Proteins Associated With Genetic Risk Of Abdominal Aortic Aneurysm: The Atherosclerosis Risk In Communities Study (ARIC)

Introduction: Abdominal aortic aneurysm (AAA) is a condition characterized by a weakened dilated vessel wall with the potential for lethal rupture. A recent genome-wide association study identified and replicated nine AAA-related variants (rs602633, rs4129267, rs3827066, rs1795061, rs10757274, rs10985349, rs9316871, rs6511720, and rs2836411), but pathways through which these loci may influence disease have not been elucidated. Hypothesis: AAA risk variants were hypothesized to be associated with plasma proteins with numerous roles including, but not limited to, inflammation and extracellular matrix remodeling. Methods: Data from participants of the community-based ARIC Study were used. Genomic DNA from whole blood was genotyped using the Affymetrix Genome-Wide Human SNP array 6.0. Concentrations of 4,870 proteins were determined using the SomaLogic aptamer-based capture array in plasma collected at visit 3 (1993-95). Outliers that were 6 standard deviations from the means were excluded. Race-specific multiple linear regression analysis evaluated associations between genetic variants and log base 2 transformed protein levels, with adjustment for age, sex, estimated glomerular filtration rate, field center, and ten principal components of ancestry. Identified proteins in whites (N=7,241) were then examined for replication in Black participants (N=1,671). Cox regression was used to evaluate the associations between identified proteins and incident AAA (n=454) over a median 21.2-year follow-up in all 11,064 participants. Results: In white participants twenty-six protein associations were identified for rs602633 ( PSRC1-CELSR2-SORT1 ), rs4129267 ( IL6R ), and rs3827066 ( PCIF1-ZNF335-MMP9 ) following Bonferroni correction ( p ≤5.13x10 -6 ). Associations were observed between rs4129267 and soluble interleukin-6 receptor subunit alpha (β=-0.339; p<1.0E-200) and CRP (β=0.093; p=2.24E-7); rs602633 and granulin (β=0.184; p<1.0E-200), complement C1q TNF-related protein-1 (β=0.115; p=2.17E-184), and neogenin (β=-0.031; p=5.60E-13); and rs3827066 and kit ligand (β=-0.045; p=2.41E-08). Five associations were replicated in Black participants ( p ≤1.9x10 -3 ). In the whole cohort, top quintiles of CRP (HR: 1.68; 95% CI: 1.22, 2.31), kit ligand (HR: 0.56; 95% CI: 0.40, 0.78) and neogenin (HR: 0.57 95% CI: 0.42, 0.78) were significantly related to incident AAA risk compared to corresponding bottom quintiles. Conclusions: Three of the nine AAA risk variants were associated with plasma protein concentrations, with the strongest associations for those involved in inflammation, endothelial dysfunction, and extracellular matrix remodeling. Granulin, complement C1q tumor necrosis factor-related protein-1, kit ligand, and neogenin represent novel targets for genetic risk of AAA and may link genetic susceptibility to disease pathogenesis.

The Effect of Bone Marrow Transplantation on Oocyte-Granulosa Cell Interaction and Follicular Development of Cisplatin-Induced Ovarian Failure in Rat

Introduction: Chemotherapy has cytotoxic effect that induces follicular damage and abnormal folliculogenesis leads to ovarian failure. Two crucial growth factors in abnormal folliculogenesis, Growth Differentiation Factor-9 (GDF-9) and Kit-Ligand, will be disrupted and affect follicular development. In this study, we evaluate whether bone marrow transplantation (BMT) has a role on oocyte-granulosa cell interaction by analyzing GDF-9 and Kit-Ligand expressions and also follicular development by analyzing primordial, primary, secondary and graafian follicles of cisplatin-induced ovarian failure in rat. Material and method: Forty eight rats were divided into three groups: control, cisplatin and cisplatin + BMT. Ovarian failure was induced by administration of intraperitoneal cisplatin 5 mg/kg body weight for 1 week. BMT 2 x107 cells were injected through rat tail vein after cisplatin administration. Bone marrow was isolated from rat femur and characterized by CD44(+), CD45(-), CD105(+). Immunohistochemistry examinations for ovarian GDF-9, Kit-Ligand and follicle development evaluation were performed after 2 weeks of BMT injection. Results: The expressions of Kit-ligand among three groups by ANOVA were significant different (p=0.00), whereas by Post Hoc: cisplatin group lower than control group (p=0.00); cisplatin + BMT group higher than cisplatin group (p=0.00); and no significant different between control group and cisplatin + BMT group (p=0.955). The expressions of GDF-9 by Kruskal Wallis showed significant different (p=0.00) among three groups whereas cisplatin + BMT group higher than cisplatin group and control group. In cisplatin + BMT group the number of primordial, primary, secondary and graafian follicles were higher than those in cisplatin group; but were lower than those in control group (p=0.000). Positive Paul Kart Horan (PKH) labeling was seen in cisplatin + BMT group only. Conclusion: In cisplatin-induced ovarian failure in rat, bone marrow transplantation may improve oocytegranulosa cell interaction and follicular development.

Regulation of Folliculogenesis by Growth Factors in Piglet Ovary Exposed Prenatally to β-Hydroxy-β-Methylbutyrate (HMB)

AbstractΒ-hydroxy-β-methylbutyrate (HMB) is one of the leucine metabolites with protein anabolic effects which makes it very popular among athletes. Previously, it was shown that HMB administered during the prenatal period reduced the pool of primordial follicles and increased the proportion of developing follicles in newborn piglets. This work is a further step to understand these morphological alterations. Therefore, the aim of this study was to examine the effect of prenatal HMB treatment on the expression of the Kit ligand, BMP-4, bFGF, and the IGF-1/IGF-1R system which are the main growth factors controlling follicular development. Excised ovaries from 12 newborn piglets, originated from the control (n=6) and HMB-treated (n=6) sows were used for immunohistochemical and western-blot analysis. The tested proteins were localized within egg nests and ovarian follicles. Furthermore, the western-blot assay indicated higher BMP-4, Kit ligand, and IGF-1R expression, while the level of bFGF and IGF-1 proteins decreased after HMB dietary treatment. These findings show that HMB included into sow diet can modulate the expression of growth factors and thereby alter ovarian morphology in offspring. Therefore, this study opens a discussion about the benefits and risks of the diet supplemented with HMB and its potential application in medicine and animal husbandry, and further research is necessary in this area.

Identification of a Novel Mutation in the KITLG Gene in a Chinese Family with Familial Progressive Hyper- and Hypopigmentation

Background Familial progressive hyper- and hypopigmentation (FPHH, MIM 145250) is a rare hereditary skin disorder that is predominantly characterized by progressive, diffuse, partly blotchy hyperpigmented lesions intermingled with scattered hypopigmented spots, lentigines and sometimes Cafe-au-lait spots (CALs). Heterozygous mutations of KIT ligand (KITLG, MIM 184745) gene, which encodes KIT ligand protein, is responsible for FPHH.Results A novel mutation c.104A > T (p.Asn35Ile) and a recurrent mutation c.101C > T (p.Thr34Ile) in the KITLG gene with two Chinese FPHH families were identified. So far, various pathogenic gain-of-function mutations in the KITLG gene have been described, which are located in or near the conserved VTNN motif (amino acid 33–37) in exon 2 of the KITLG gene. The reported mutations are only involved in 33V, 34T, 36N, 37V but not 35N. As SIFT and Polyphen-2 softwares showed, these two mutations were both predicted to be detrimental variations. Three-dimensional protein structures modeling indicated the mutant KITLG proteins might affect KITLG affinity to its receptor c-KIT. To date, only eight KITLG mutations were associated with FPHH and no clear genotype-phenotype correlations had been well established.Conclusions We have now identified a novel mutation c.104A > T (p.Asn35Ile) of KITLG gene, which was first reported in FPHH located within the conserved 35N of the motif. These results strengthen our understanding of FPHH and expand the mutational spectrum of the KITLG gene.

Lung Function in African American Children with Asthma Is Associated with Novel Regulatory Variants of the KIT Ligand KITLG/SCF and Gene-By-Air-Pollution Interaction

Baseline lung function, quantified as forced expiratory volume in the first second of exhalation (FEV1), is a standard diagnostic criterion used by clinicians to identify and classify lung diseases. Using whole-genome sequencing data from the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine project, we identified a novel genetic association with FEV1 on chromosome 12 in 867 African American children with asthma (P = 1.26 × 10−8, β = 0.302). Conditional analysis within 1 Mb of the tag signal (rs73429450) yielded one major and two other weaker independent signals within this peak. We explored statistical and functional evidence for all variants in linkage disequilibrium with the three independent signals and yielded nine variants as the most likely candidates responsible for the association with FEV1. Hi-C data and expression QTL analysis demonstrated that these variants physically interacted with KITLG (KIT ligand, also known as SCF), and their minor alleles were associated with increased expression of the KITLG gene in nasal epithelial cells. Gene-by-air-pollution interaction analysis found that the candidate variant rs58475486 interacted with past-year ambient sulfur dioxide exposure (P = 0.003, β = 0.32). This study identified a novel protective genetic association with FEV1, possibly mediated through KITLG, in African American children with asthma. This is the first study that has identified a genetic association between lung function and KITLG, which has established a role in orchestrating allergic inflammation in asthma.

KIT ligand protects against both light-induced and genetic photoreceptor degeneration

Photoreceptor degeneration is a major cause of blindness and a considerable health burden during aging but effective therapeutic or preventive strategies have not so far become readily available. Here, we show in mouse models that signaling through the tyrosine kinase receptor KIT protects photoreceptor cells against both light-induced and inherited retinal degeneration. Upon light damage, photoreceptor cells upregulate Kit ligand (KITL) and activate KIT signaling, which in turn induces nuclear accumulation of the transcription factor NRF2 and stimulates the expression of the antioxidant gene Hmox1. Conversely, a viable Kit mutation promotes light-induced photoreceptor damage, which is reversed by experimental expression of Hmox1. Furthermore, overexpression of KITL from a viral AAV8 vector prevents photoreceptor cell death and partially restores retinal function after light damage or in genetic models of human retinitis pigmentosa. Hence, application of KITL may provide a novel therapeutic avenue for prevention or treatment of retinal degenerative diseases.