Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Transcription factors in fibroblast plasticity and CAF heterogeneity.

In recent years, research focused on the multifaceted landscape and functions of cancer-associated fibroblasts (CAFs) aimed to reveal their heterogeneity and identify commonalities across diverse tumors for more effective therapeutic targeting of pro-tumoral stromal microenvironment. However, a unified functional categorization of CAF subsets remains elusive, posing challenges for the development of targeted CAF therapies in clinical settings.The CAF phenotype arises from a complex interplay of signals within the tumor microenvironment, where transcription factors serve as central mediators of various cellular pathways. Recent advances in single-cell RNA sequencing technology have emphasized the role of transcription factors in the conversion of normal fibroblasts to distinct CAF subtypes across various cancer types.This review provides a comprehensive overview of the specific roles of transcription factor networks in shaping CAF heterogeneity, plasticity, and functionality. Beginning with their influence on fibroblast homeostasis and reprogramming during wound healing and fibrosis, it delves into the emerging insights into transcription factor regulatory networks. Understanding these mechanisms not only enables a more precise characterization of CAF subsets but also sheds light on the early regulatory processes governing CAF heterogeneity and functionality. Ultimately, this knowledge may unveil novel therapeutic targets for cancer treatment, addressing the existing challenges of stromal-targeted therapies.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo.

Nucleophosmin (NPM), a nucleolar multifunctional phosphoprotein, acts as a stress sensor in different cell types. NPM can be actively secreted by inflammatory cells, however its biology on endothelium remains unexplored. In this study, we show for the first time that NPM is secreted by human vein endothelial cells (HUVEC) in the early response to serum deprivation and that NPM acts as a pro-inflammatory and angiogenic molecule both in vitro and in vivo. Accordingly, 24 h of serum starvation condition induced NPM relocalization from the nucleus to cytoplasm. Interestingly, NPM was increasingly excreted in HUVEC-derived conditioned media in a time dependent fashion upon stress conditions up to 24 h. The secretion of NPM was unrelated to cell necrosis within 24 h. The treatment with exogenous and recombinant NPM (rNPM) enhanced migration as well as the Intercellular Adhesion Molecule 1 (ICAM-1) but not Vascular cell adhesion protein 1 (VCAM-1) expression and it did not affect cell proliferation. Notably, in vitro tube formation by Matrigel assay was significantly increased in HUVEC treated with rNPM compared to controls. This result was confirmed by the in vivo injection of Matrigel plug assay upon stimulation with rNPM, displaying significant enhanced number of functional capillaries in the plugs. The stimulation with rNPM in HUVEC was also associated to the increased expression of master genes regulating angiogenesis and migration, including Vascular Endothelial Growth Factor-A (VEGF-A), Hepatocyte Growth Factor (HGF), Stromal derived factor-1 (SDF-1), Fibroblast growth factor-2 (FGF-2), Platelet Derived Growth Factor-B (PDGF-B), and Matrix metallopeptidase 9 (MMP9). Our study demonstrates for the first time that NPM is physiologically secreted by somatic cells under stress condition and in the absence of cell necrosis. The analysis of the biological effects induced by NPM mainly related to a pro-angiogenic and inflammatory activity might suggest an important autocrine/paracrine role for NPM in the regulation of both phenomena.

The actin modulator hMENA regulates GAS6-AXL axis and pro-tumor cancer/stromal cell cooperation.

The dynamic interplay between cancer cells and cancer-associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue-specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor-promoting CAFs and in the modulation of pro-tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC-MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL-expressing pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6-AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor-stroma crosstalk, with far-reaching prognostic and therapeutic implications for NSCLC and PDAC.

Successful recovery from COVID-19 pneumonia after receiving baricitinib, tocilizumab, and remdesivir. A case report: Review of treatments and clinical role of computed tomography analysis.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic, threatening global public health. In the current paper, we describe our successful treatment of one COVID-19 pneumonia patient case with high mortality risk factors. Our experience underlines the importance of the use of a multidisciplinary therapeutic approach in order to achieve a favorable clinical outcome. Further, enhancing the capability of the COVID-19 diagnosis with the use of the chest imaging modalities is discussed.

hMENA isoforms impact NSCLC patient outcome through fibronectin/ß1 integrin axis.

We demonstrated previously that the splicing of the actin regulator, hMENA, generates two alternatively expressed isoforms, hMENA11a and hMENAΔv6, which have opposite functions in cell invasiveness. Their mechanisms of action have remained unclear. Here we report two major findings: (i) hMENA regulates ß1 integrin expression. This was shown by depleting total hMENA, which led to loss of nuclear expression of serum response factor (SRF)-coactivator myocardin-related transcription factor 1 (MRTF-A), leading to an increase in the G-actin/F-actin ratio crucial for MRTF-A localization. This in turn inhibited SRF activity and the expression of its target gene ß1 integrin. (ii) hMENA11a reduces and hMENAΔv6 increases ß1 integrin activation and signaling. Moreover, exogenous expression of hMENA11a in hMENAΔv6-positive cancer cells dramatically reduces secretion of extracellular matrix (ECM) components, including ß1 integrin ligands and metalloproteinases. On the other hand, overexpression of the pro-invasive hMENAΔv6 increases fibronectin production. In primary tumors high hMENA11a correlates with low stromal fibronectin and a favorable clinical outcome of early node-negative non-small-cell lung cancer patients. These data provide new insights into the roles of hMENA11a and hMENAΔv6 in the druggable ß1 integrin-ECM signaling axis and allow stratification of patient risk, guiding their clinical management.

Naturally Adipose Stromal Cell-Enriched Fat Graft: Comparative Polychromatic Flow Cytometry Study of Fat Harvested by Barbed or Blunt Multihole Cannula.

Background: Fat grafts enriched with cells of the stromal vascular fraction (SVF), especially adipose-derived stromal cells (ASCs), exhibit significantly improved retention over non enriched, plain fat. Different types of liposuction cannulae may yield lipoaspirates with different subpopulations of cells. Moreover, preparation of adipose tissue for transplantation typically involves centrifugation, which creates a density gradient of fat. Objectives: The authors sought to determine whether liposuction with a barbed or smooth cannula altered the enrichment of the SVF, and specifically ASCs, in low-density (LD) and high-density (HD) fractions of centrifuged adipose tissue. Methods: Fat was harvested from 2 abdominal sites of 5 healthy women with a barbed or smooth multihole blunt-end cannula. After centrifugation, LD and HD fat fractions were digested with collagenase and analyzed by polychromatic flow cytometry to identify and enumerate distinct populations of cells. Results: Overall cell yield and the number of immune cells were consistently higher in HD fractions than in LD fractions, regardless of the cannula employed. More living cells, and specifically more ASCs, populated the HD fractions of lipoaspirates obtained with a barbed cannula than with a smooth cannula. Conclusions: In this study, lipoaspiration with a barbed cannula and isolation of the HD layer of centrifuged adipose tissue yielded maximal amounts of SVF cells, including ASCs.

Hematopoietic activity in putative mouse primordial germ cell populations.

In the present paper, starting from the observation of heterogeneous expression of the GOF-18ΔPE-GFP Pou5f1 (Oct3/4) transgene in putative mouse PGC populations settled in the aorta-gonad-mesonephros (AGM) region, we identified various OCT3/4 positive populations showing distinct expression of PGC markers (BLIMP-1, AP, TG-1, STELLA) and co-expressing several proteins (CD-34, CD-41, FLK-1) and genes (Brachyury, Hox-B4, Scl/Tal-1 and Gata-2) of hematopoietic precursors. Moreover, we found that Oct3/4-GFP(weak) CD-34(weak/high) cells possess robust hematopoietic colony forming activity (CFU) in vitro. These data indicate that the cell population usually considered PGCs moving toward the gonadal ridges encompasses a subset of cells co-expressing several germ cell and hematopoietic markers and possessing hematopoietic activity. These results are discussed within of the current model of germline segregation.

Transcriptional profiling of HMGB1-induced myocardial repair identifies a key role for Notch signaling.

Exogenous high-mobility group box 1 protein (HMGB1) administration to the mouse heart, during acute myocardial infarction (MI), results in cardiac regeneration via resident c-kit(+) cell (CPC) activation. Aim of the present study was to identify the molecular pathways involved in HMGB1-induced heart repair. Gene expression profiling was performed to identify differentially expressed genes in the infarcted and bordering regions of untreated and HMGB1-treated mouse hearts, 3 days after MI. Functional categorization of the transcripts, accomplished using Ingenuity Pathway Analysis software (IPA), revealed that genes involved in tissue regeneration, that is, cardiogenesis, vasculogenesis and angiogenesis, were present both in the infarcted area and in the peri-infarct zone; HMGB1 treatment further increased the expression of these genes. IPA revealed the involvement of Notch signaling pathways in HMGB1-treated hearts. Importantly, HMGB1 determined a 35 and 58% increase in cardiomyocytes and CPCs expressing Notch intracellular cytoplasmic domain, respectively. Further, Notch inhibition by systemic treatment with the γ-secretase inhibitor DAPT, which blocked the proteolytic activation of Notch receptors, reduced the number of CPCs, their proliferative fraction, and cardiomyogenic differentiation in HMGB1-treated infarcted hearts. The present study gives insight into the molecular processes involved in HMGB1-mediated cardiac regeneration and indicates Notch signaling as a key player.

C/EBPγ regulates wound repair and EGF receptor signaling.

We aimed at identifying novel regulators of skin wound healing (WH), in an epidermal scratch WH assay, by a small interfering RNA (siRNA) silencing approach. Several transcription factors have been previously reported to affect wound repair. We here show that gene silencing of the transcription factor CAAT enhancer-binding protein γ (C/EBPγ), STAT3, REL, RELA, RELB, SP1, and NFkB impaired WH in vitro, in keratinocytes, whereas E2F and CREBBP silencing accelerated the WH process. We further characterized C/EBPγ, as its silencing yielded the maximal impairment (52.2 ± 12.5%) of scratch wounding (SW). We found that C/EBPγ silencing inhibited both EGF- and serum-induced keratinocyte migration, whereas C/EBPγ overexpression enhanced cell migration to EGF and to serum via the EGFR. Further, C/EBPγ silencing impaired scratch-induced Y1068 and Y1173 EGFR phosphorylation, as well as Y118 paxillin phosphorylation, key molecules regulating cell migration and epidermal WH. Moreover, C/EBPγ levels were induced in keratinocytes, following both SW and EGF stimulation. C/EBPγ siRNA silencing in vivo impaired WH at 3, 5, 7, and 14 days following excisional wounding in mice inhibited both re-epithelialization and granulation tissue formation, and induced a decrease of arteriole number. In conclusion, we here report that C/EBPγ positively regulates wound repair both in vitro and in vivo, at least in part, by affecting EGFR signaling.

HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3.

AIMS: HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts. METHODS AND RESULTS: Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit⁺ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20-25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206. CONCLUSIONS: HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.

Circulating microRNAs are new and sensitive biomarkers of myocardial infarction.

AIMS: Circulating microRNAs (miRNAs) may represent a novel class of biomarkers; therefore, we examined whether acute myocardial infarction (MI) modulates miRNAs plasma levels in humans and mice. METHODS AND RESULTS: Healthy donors (n = 17) and patients (n = 33) with acute ST-segment elevation MI (STEMI) were evaluated. In one cohort (n = 25), the first plasma sample was obtained 517 ± 309 min after the onset of MI symptoms and after coronary reperfusion with percutaneous coronary intervention (PCI); miR-1, -133a, -133b, and -499-5p were ~15- to 140-fold control, whereas miR-122 and -375 were ~87-90% lower than control; 5 days later, miR-1, -133a, -133b, -499-5p, and -375 were back to baseline, whereas miR-122 remained lower than control through Day 30. In additional patients (n = 8; four treated with thrombolysis and four with PCI), miRNAs and troponin I (TnI) were quantified simultaneously starting 156 ± 72 min after the onset of symptoms and at different times thereafter. Peak miR-1, -133a, and -133b expression and TnI level occurred at a similar time, whereas miR-499-5p exhibited a slower time course. In mice, miRNAs plasma levels and TnI were measured 15 min after coronary ligation and at different times thereafter. The behaviour of miR-1, -133a, -133b, and -499-5p was similar to STEMI patients; further, reciprocal changes in the expression levels of these miRNAs were found in cardiac tissue 3-6 h after coronary ligation. In contrast, miR-122 and -375 exhibited minor changes and no significant modulation. In mice with acute hind-limb ischaemia, there was no increase in the plasma level of the above miRNAs. CONCLUSION: Acute MI up-regulated miR-1, -133a, -133b, and -499-5p plasma levels, both in humans and mice, whereas miR-122 and -375 were lower than control only in STEMI patients. These miRNAs represent novel biomarkers of cardiac damage.

Induction of myogenic differentiation by SDF-1 via CXCR4 and CXCR7 receptors.

The stromal cell-derived factor (SDF)-1/CXC receptor 4 (CXCR4) axis has been shown to play a role in skeletal muscle development, but its contribution to postnatal myogenesis and the role of the alternate SDF-1 receptor, CXC receptor 7 (CXCR7), are poorly characterized. Western blot analysis and real-time polymerase chain reaction (PCR) were performed to evaluate in vitro the effect of SDF-1 and CXCR4 and CXCR7 inhibition on myogenic differentiation. Proliferating myoblasts express CXCR4, CXCR7, and SDF-1; during myogenic differentiation, CXCR4 and CXCR7 levels are downregulated, and SDF-1 release is decreased. SDF-1 anticipates myosin heavy chain accumulation and myotube formation in both C2C12 myoblasts and satellite cells. Interestingly, inhibition of CXCR4 and CXCR7 signaling, either by drugs or RNA interfererence, blocks myogenic differentiation. Further, the CXCR4 antagonist, 4F-benzoyl-TN14003, inhibits myoblast cell cycle withdrawal and decreases the retinoblastoma gene (pRb) product accumulation in its hypophosphorylated form. Our experiments demonstrate that SDF-1 regulates myogenic differentiation via both CXCR4 and CXCR7 chemokine receptors.

Myocardial infarction induces embryonic reprogramming of epicardial c-kit(+) cells: role of the pericardial fluid.

Stem cells expressing c-kit have been identified in the adult epicardium. In mice, after myocardial infarction, these cells proliferate, migrate to the injury site and differentiate toward myocardial and vascular phenotype. We hypothesized that, acutely after myocardial infarction, pericardial sac integrity and pericardial fluid (PF) may play a role on epicardial cell gene expression, proliferation and differentiation. Microarray analysis indicated that, in the presence of an intact pericardial sac, myocardial infarction modulated 246 genes in epicardial cells most of which were related to cell proliferation, cytoskeletal organization, wound repair and signal transduction. Interestingly, WT1, Tbx18 and RALDH2, notably involved in epicardial embryonic development, were markedly up-regulated. Importantly, coexpression of stem cell antigen c-kit and WT1 and/or Tbx18 was detected by immunohistochemistry in the mouse epicardium during embryogenesis as well as in adult mouse infarcted heart. Injection of human pericardial fluid from patients with acute myocardial ischemia (PFMI) in the pericardial cavity of non-infarcted mouse hearts, enhanced, epicardial cell proliferation and WT1 expression. Further, PFMI supplementation to hypoxic cultured human epicardial c-kit(+) cells increased WT1 and Tbx18 mRNA expression. Finally, insulin-like growth factor 1, hepatocyte growth factor and high mobility group box 1 protein, previously involved in cardiac c-kit(+) cell proliferation and differentiation, were increased in PFMI compared to the pericardial fluid of non ischemic patients. In conclusion, myocardial infarction reactivates an embryonic program in epicardial c-kit(+) cells; soluble factors released in the pericardial fluids following myocardial necrosis may play a role in this process.

High-mobility group box 1 protein in human and murine skin: involvement in wound healing.

High-mobility group box 1 (HMGB1) protein is a multifunctional cytokine involved in inflammatory responses and tissue repair. In this study, it was examined whether HMGB1 plays a role in skin wound repair both in normoglycemic and diabetic mice. HMGB1 was detected in the nucleus of skin cells, and accumulated in the cytoplasm of epidermal cells in the wounded skin. Diabetic human and mouse skin showed more reduced HMGB1 levels than their normoglycemic counterparts. Topical application of HMGB1 to the wounds of diabetic mice enhanced arteriole density, granulation tissue deposition, and accelerated wound healing. In contrast, HMGB1 had no effect in normoglycemic mouse skin wounds, where endogenous HMGB1 levels may be adequate for optimal wound closure. Accordingly, inhibition of endogenous HMGB1 impaired wound healing in normal mice but had no effect in diabetic mice. Finally, HMGB1 had a chemotactic effect on skin fibroblasts and keratinoyctes in vitro. In conclusion, lower HMGB1 levels in diabetic skin may play an important role in impaired wound healing and this defect may be overcome by the topical application of HMGB1.

Multiple effects of high mobility group box protein 1 in skeletal muscle regeneration.

OBJECTIVE: High mobility group box 1 protein (HMGB1) is a cytokine released by necrotic and inflammatory cells in response to injury. We examined the role of HMGB1 in skeletal muscle regeneration after hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced in mice by femoral artery dissection. HMGB1 levels increased in regenerating skeletal muscle and the blockade of endogenous HMGB1 by the administration of its truncated form, the BoxA, resulted in the reduction of vessel density. In contrast, intramuscular administration of HMGB1 enhanced perfusion and increased the number of regenerating fibers. To separately study the myogenic and the angiogenic effects of HMGB1, in vitro experiments were performed with isolated myoblasts and endothelial cells. Myoblasts were found to express the HMGB1 receptor RAGE and TLR4 which were downregulated during in vitro myogenic differentiation. HMGB1 was extracellularly released by differentiated myoblasts and exerted a chemotactic activity on myogenic cells. This effect was partially dependent on RAGE and was inhibited by BoxA treatment. Finally, HMGB1 stimulated tubular-like structure formation by endothelial cells through the activation of extracellular signal-regulated kinase (ERK) and JNK signal transduction pathways. CONCLUSIONS: HMGB1 plays a role in skeletal muscle regeneration modulating, in an autocrine-paracrine manner, myoblast and endothelial cell functions.

Exogenous high-mobility group box 1 protein induces myocardial regeneration after infarction via enhanced cardiac C-kit+ cell proliferation and differentiation.

High-mobility group box 1 protein (HMGB1) is a chromatin protein that is released by inflammatory and necrotic cells. Extracellular HMGB1 signals tissue damage, stimulates the secretion of proinflammatory cytokines and chemokines, and modulates stem cell function. The present study examined exogenous HMGB1 effect on mouse left-ventricular function and myocyte regeneration after infarction. Myocardial infarction was induced in C57BL/6 mice by permanent coronary artery ligation. After 4 hours animals were reoperated and 200 ng of purified HMGB1 was administered in the peri-infarcted left ventricle. This intervention resulted in the formation of new myocytes within the infarcted portion of the wall. The regenerative process involved the proliferation and differentiation of endogenous cardiac c-kit+ progenitor cells. Circulating c-kit+ cells did not significantly contribute to HMGB1-mediated cardiac regeneration. Echocardiographic and hemodynamic parameters at 1, 2, and 4 weeks demonstrated a significant recovery of cardiac performance in HMGB1-treated mice. These effects were not observed in infarcted hearts treated either with the unrelated protein glutathione S-transferase or a truncated form of HMGB1. Thus, HMGB1 appears to be a potent inducer of myocardial regeneration following myocardial infarction.

Enhanced arteriogenesis and wound repair in dystrophin-deficient mdx mice.

BACKGROUND: The absence of functional dystrophin in Duchenne muscular dystrophy (DMD) patients and in mdx mice results in progressive muscle degeneration associated with necrosis, fibrosis, and inflammation. Because vascular supply plays a key role in tissue repair, we examined whether new blood vessel development was altered in mdx mice. METHODS AND RESULTS: In a model of hindlimb ischemia on femoral artery dissection, hindlimb perfusion, measured by laser Doppler imaging, was higher in mdx mice (0.67+/-0.26) than in wild-type (WT) mice (0.33+/-0.18, P<0.03). In keeping with these data, a significant increase in arteriole length density was found in mdx mice (13.6+/-8.4 mm/mm3) compared with WT mice (7.8+/-4.6 mm/mm3, P<0.03). Conversely, no difference was observed in capillary density between mice of the 2 genotypes. The enhanced regenerative response was not limited to ischemic skeletal muscle, because in a wound-healing assay, mdx mice showed an accelerated wound closure rate compared with WT mice. Moreover, a vascularization assay in Matrigel plugs containing basic fibroblast growth factor injected subcutaneously revealed an increased length density of arterioles in mdx (46.9+/-14.7 mm/mm3) versus WT mice (19.5+/-5.8 mm/mm3, P<0.001). Finally, serum derived from mdx mice sustained formation of endothelium-derived tubular structures in vitro more efficiently than WT serum. CONCLUSIONS: These results demonstrate that arteriogenesis is enhanced in mdx mice both after ischemia and skin wounding and in response to growth factors.

Meiotic progression of isolated mouse spermatocytes under simulated microgravity.

Progression through the prophase of the first meiotic division can be obtained in culture by treatment of mouse spermatocytes with the serine/threonine phosphatase inhibitor okadaic acid. Chromosome condensation during this G2/M transition involves the activation of the MAPK pathway, which causes the activation of Nek2 and the phosphorylation of the chromatin architectural protein Hmga2. In an effort to set up conditions to allow a spontaneous progression of mouse spermatocytes through meiosis, we have investigated the cell-cycle features of these cells cultured for 24 h with a rotary cell culture system in a humidified atmosphere in a thermostatic incubator to simulate a microgravity environment. Morphological analysis of nuclear squashes indicated a 2-fold increase in late-pachytene spermatocytes with highly condensed chromosomes, and a contemporaneous decrease of mid-pachytene cells with less condensed chromatin. Microgravity induced a 2-fold activation of the cyclinB-cdc2 complex, confirming at the molecular level that cell-cycle progression had occurred. Moreover, using immuno-kinase assays with specific substrates we have demonstrated that the meiotic progression obtained under microgravity conditions is accompanied by activation of the Erk1/p90Rsk2 pathway. These data indicated that activation of the MAPK pathway correlates with chromatin condensation even under conditions in which meiotic progression occurs spontaneously and is not induced by a drug. We suggest that culture under microgravity conditions might help to release the block that inhibits isolated spermatocytes from progressing through prophase at unit gravity, and to study the physiological events of germ cell differentiation in vitro.