Post-traumatic heterotopic ossification (HO) is the formation of ectopic bone in non-osseous structures following injury. The precise mechanism for bone development following trauma is unknown; however, early onset of HO may involve the production of pro-osteogenic serum factors. Here we evaluated serum from a cohort of civilian and military patients post trauma to determine early induction gene signatures in orthopaedic trauma induced HO. To test this, human adipose derived stromal/stem cells (hASCs) were stimulated with human serum from patients who developed HO following trauma and evaluated for a gene panel with qPCR. Pathway gene analysis ontology revealed that hASCs stimulated with serum from patients who developed HO had altered gene expression in the activator protein 1 (AP1) and AP1 transcriptional targets pathways. Notably, there was a significant repression in FOS gene expression in hASCs treated with serum from individuals with HO. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway was activated in hASCs following serum exposure from individuals with HO. Serum from both military and civilian patients with trauma induced HO had elevated downstream genes associated with the MAPK pathways. Stimulation of hASCs with known regulators of osteogenesis (BMP2, IL6, Forskolin, and WNT3A) failed to recapitulate the gene signature observed in hASCs following serum stimulation, suggesting non-canonical mechanisms for gene regulation in trauma induced HO. These findings provide new insight for the development of HO and support ongoing work linking the systemic response to injury with wound specific outcomes.
Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.
Abstract Cancers acquire somatic mitochondrial DNA (mtDNA) mutations undetected in surrounding tissues. Cancer-specific mtDNA mutations primarily constitute variants of unknown significance whose oncogenic consequences are poorly understood. Analysis of large-scale sequencing datasets suggests that human cancers generally select against severe mtDNA mutations; however, the presence of selective pressure on the mtDNA genome has yet to be experimentally demonstrated. Here we optimized a FACS-based protocol to reconstitute the highly metastatic human melanoma cell line A375 with exogenous mtDNA, thereby generating nuclear-isogenic cytoplasmic hybrid (cybrid) lines. Homoplasmic cybrids were generated exclusively harboring either exogenous wildtype mtDNA, mtDNA with partial loss of OXHPHOS function (SNPs in mt.ND1 (of Complex I) or mt.ATP6 (of Complex V)), or mtDNA with complete loss of OXPHOS function (derived from a human melanoma harboring a truncating frameshift mutation in mt.CO1 (of Complex IV)). Compared to wildtype cybrids, subcutaneous xenografts of ND1 and ATP6 cybrids into NOD–SCID Il2rg−/− (NSG) mice had elevated primary tumor ROS levels and similar primary tumor growth rates. Xenografts of our most dysfunctional OXPHOS cybrid, CO1, formed tumors albeit with lowered growth rates. Uniquely, the ND1 and CO1 xenografts respectively exhibited diminished metastatic burden and no metastatic burden. Our results suggest that OXPHOS impairing mtDNA mutations are tolerable during primary tumor growth but reduce the severity and incidence of metastasis. Lastly, we generated heteroplasmic A375 cybrids, harboring both wild-type and mutant alleles, and developed a ddPCR method to characterize allelic frequency with single-cell precision. Extended in vitro passage of the heteroplasmic cybrids failed to significantly shift the heteroplasmic mtDNA allelic frequency. However, following subcutaneous xenografting of the heteroplasmic cybrids, primary tumors significantly shifted their variant allele frequency to favor the wildtype allele. In summary, these results indicate that a functional mtDNA haplotype is not absolutely required for tumorigenesis but contributes to metastatic efficiency. In addition, we experimentally demonstrated the existence of selective pressure in favor of functional mtDNA during tumorigenesis. Citation Format: Spencer Shelton, Sara House, Vijayashree Ramesh, Zhengkang Chen, Claire Llamas, Alpaslan Tasdogan, Ralph DeBerardinis, Sean Morrison, Prashant Mishra. OXPHOS impairing mitochondrial DNA mutations suppress melanoma growth and metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR003.
Chronic wounds are characterized by an increased bacterial presence, alkaline pH, and excessive wound drainage. Hydrogel biomaterials composed of the carbohydrate polymer chitosan are advantageous for wound healing applications because of their innate antimicrobial and hemostatic properties. Here, genipin-cross-linked-chitosan hydrogels were synthesized and characterized, and their in vitro and in vivo performances were evaluated as a viable wound dressing. Characterization studies demonstrate that the developed chitosan-genipin hydrogels were able to neutralize an environmental pH, while averaging ∼230% aqueous solution uptake, demonstrating their use as a perfusive wound dressing. Bacterial activity studies demonstrate the hydrogels' ability to hinder Escherichia coli growth by ∼70%, while remaining biocompatible in vitro to fibroblast and keratinocyte cells. Furthermore, chitosan-genipin hydrogels promote an enhanced immune response and cellular proliferation in induced pressure wounds in mice. All together, these results reflect the potential of the developed hydrogels to be used as a proactive wound dressing.
Background: Processed microvascular tissue (PMVT), a human structural allograft, is derived from lyophilized human tissue containing microcirculatory cellular components. Since PMVT serves as a source of extracellular matrix (ECM), growth factors, cytokines, and chemokines modulating angiogenesis, inflammation, apoptosis, and endogenous cell recruitment, we hypothesized its application would accelerate wound regeneration in a validated pressure ulcer (PU) model developed in C57BL/6 mice using two 24-hour cycles of skin ischemia/reperfusion created by placement and removal of external magnets. Methods: Two identical PU injuries (n = 50 female mice) were treated with (a) topical particulate PMVT, (b) injected rehydrated PMVT, or (c) saline control injection, and assessed daily for closure rates, scab formation/removal, and temperature. A baseline control cohort (n = 5) was euthanized at day 0 and treatment group cohorts (n = 5) were killed at 3, 7, or 14 days postinjury. The PU injuries were collagenase-digested for flow cytometric analysis of inflammatory, reparative, and stem cell frequencies and analyzed by hematoxylin and eosin (H&E) histology and immunofluorescence. Results: PMVT-accelerated wound closure, most notably, topical PMVT significantly increased mean closure from d5 (13% versus -9%) through d13 (92% versus 38%) compared with phosphate-buffered saline (PBS) controls ( P < 0.05). PMVT also hastened scab formation/removal, significantly accelerated disappearance of inflammatory myeloid (CD11b+) cells while upregulating α-smooth muscle actin, vascular endothelial growth factor A, and placental growth factor and raised skin temperature surrounding the PU site, consistent with increased blood flow. Conclusions: These results indicate that PMVT has potential as an advanced treatment for restoring normal tissue function in ischemic wounds and merits clinical study.
The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression.Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice.ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.
Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here, we report a 21-year-old man of Chinese ancestry who has an autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia, and partial lipodystrophy. Analyses of exome sequencing data from the entire family revealed only 1 rare homozygous missense variant (c.86C>T; p.Pro29Leu) in TOMM7 in the proband, while the parents and 2 unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex makes up the outer membrane pore, which is responsible for importing many preproteins into the mitochondria. A proteomic comparison of mitochondria from control and proband-derived cultured fibroblasts revealed an increase in abundance of several proteins involved in oxidative phosphorylation, as well as a reduction in abundance of proteins involved in phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared with control fibroblasts. We concluded that altered mitochondrial protein import due to biallelic loss-of-function TOMM7 can cause severe growth retardation and progeroid features.
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