Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Specific miRNA and Gene Deregulation Characterize the Increased Angiogenic Remodeling of Thoracic Aneurysmatic Aortopathy in Marfan Syndrome.

Marfan syndrome (MFS) is a connective tissue disease caused by mutations in the FBN1 gene, leading to alterations in the extracellular matrix microfibril assembly and the early formation of thoracic aorta aneurysms (TAAs). Non-genetic TAAs share many clinico-pathological aspects with MFS and deregulation of some microRNAs (miRNAs) has been demonstrated to be involved in the progression of TAA. In this study, 40 patients undergoing elective ascending aorta surgery were enrolled to compare TAA histomorphological features, miRNA profile and related target genes in order to find specific alterations that may explain the earlier and more severe clinical outcomes in MFS patients. Histomorphological, ultrastructural and in vitro studies were performed in order to compare aortic wall features of MFS and non-MFS TAA. MFS displayed greater glycosaminoglycan accumulation and loss/fragmentation of elastic fibers compared to non-MFS TAA. Immunohistochemistry revealed increased CD133+ angiogenic remodeling, greater MMP-2 expression, inflammation and smooth muscle cell (SMC) turnover in MFS TAA. Cultured SMCs from MFS confirmed higher turnover and α-smooth muscle actin expression compared with non-MFS TAA. Moreover, twenty-five miRNAs, including miR-26a, miR-29, miR-143 and miR-145, were found to be downregulated and only miR-632 was upregulated in MFS TAA in vivo. Bioinformatics analysis revealed that some deregulated miRNAs in MFS TAA are implicated in cell proliferation, extracellular matrix structure/function and TGFß signaling. Finally, gene analysis showed 28 upregulated and seven downregulated genes in MFS TAA, some of them belonging to the CDH1/APC and CCNA2/TP53 signaling pathways. Specific miRNA and gene deregulation characterized the aortopathy of MFS and this was associated with increased angiogenic remodeling, likely favoring the early and more severe clinical outcomes, compared to non-MFS TAA. Our findings provide new insights concerning the pathogenetic mechanisms of MFS TAA; further investigation is needed to confirm if these newly identified specific deregulated miRNAs may represent potential therapeutic targets to counteract the rapid progression of MFS aortopathy.

Clusterin exerts a cytoprotective and antioxidant effect in human osteoarthritic cartilage.

Osteoarthritis (OA) is the most common joint disease characterized by destruction of articular cartilage. OA-induced cartilage degeneration causes inflammation, oxidative stress and the hypertrophic shift of quiescent chondrocytes. Clusterin (CLU) is a ubiquitous glycoprotein implicated in many cellular processes and its upregulation has been recently reported in OA cartilage. However, the specific role of CLU in OA cartilage injury has not been investigated yet. We analyzed CLU expression in human articular cartilage in vivo and in cartilage-derived chondrocytes in vitro. CLU knockdown in OA chondrocytes was also performed and its effect on proliferation, hypertrophic phenotype, apoptosis, inflammation and oxidative stress was investigated. CLU expression was upregulated in human OA cartilage and in cultured OA cartilage-derived chondrocytes compared with control group. CLU knockdown reduced cell proliferation and increased hypertrophic phenotype as well as apoptotic death. CLU-silenced OA chondrocytes showed higher MMP13 and COL10A1 as well as greater TNF-α, Nox4 and ROS levels. Our results indicate a possible cytoprotective role of CLU in OA chondrocytes promoting cell survival by its anti-apoptotic, anti-inflammatory and antioxidant properties and counteracting the hypertrophic phenotypic shift. Further studies are needed to deepen the role of CLU in order to identify a new potential therapeutic target for OA.

Adenosquamous carcinoma of the right colon with diffuse signet-ring mucinous component.

Colorectal carcinoma is one of the most frequent human malignant neoplasms. Besides the most common histological types, colorectal adenosquamous carcinoma is very rare, being microscopically constituted by tumoural glandular and squamous components. The diagnosis of primary colon adenosquamous carcinoma requires the exclusion of a metastatic squamous cell carcinoma and the extension of a primary squamous cell carcinoma from the anal-rectum junction. We report a case of primary adenosquamous carcinoma of the ascending colon in a 62-year-old Caucasian man affected by long-standing ulcerative colitis. Peritumoural lymph nodes and distant metastasis were present. We reported also a diffuse signet-ring mucinous component, which has not been previously observed. Moreover, we investigated microsatellite and all-RAS/B-RAF status, p16 and p53 protein expression, and the molecular presence of human papillomavirus DNA in all the morphologically distinct components, in order to suggest pathogenetic factors influencing the aggressive prognosis of colon adenosquamous carcinoma.

Clinical Utility of Plasma KRAS, NRAS and BRAF Mutational Analysis with Real Time PCR in Metastatic Colorectal Cancer Patients-The Importance of Tissue/Plasma Discordant Cases.

BACKGROUND: Tumor tissue (T) mutational analysis represents the standard for metastatic colorectal cancer (mCRC); however, circulating tumor DNA (ctDNA) detected by liquid biopsy in plasma (PL) can better represent tumor heterogeneity. METHODS: mCRC patients undergoing standard first-line chemotherapy with known T-KRAS/NRAS/BRAF status were enrolled in the present prospective study. PL mutations were assessed within 2 weeks before chemotherapy start with real time PCR and correlated with T status and Progression free survival (PFS). Clinical and biochemical variables including also total number of tumor lesions (TNL) and the sum of maximum diameter (SMD) of all lesions were assessed as potential predictors of T/PL discordance. RESULTS: Among 45 enrolled patients, all BRAF mutations were concordant between T and PL and there were 20% of patients RAS discordant: 9% wild type in T and mutated in PL and 11% mutated in T and wild type in PL. T mutations were significantly associated to median PFS (mPFS of 4.5, 8.3 and 22.9 months for T-BRAF mutated, T-RAS mutated, and T-wild type patients, respectively, p for trend 0.00014). PL mutations further refined prognosis: RAS wild type in T and mutated in PL had significantly shorter PFS than concordant RAS wild type in T and PL: mPFS 9.6 vs. 23.3 months, respectively, p = 0.02. Patients RAS mutated in T and wild type in PL had longer PFS than concordant RAS mutated in T and PL: 24.4 vs. 7.8 months, respectively, p = 0.008. At a multivariate cox regression analysis for PFS, PL mutations were independent prognostic factor superior to T analysis (HR 0.13, p = 0.0008). At multivariate logistic regression analysis TNL and SMD were significant predictors of discordant cases. CONCLUSIONS: PL mutational analysis allows a better prognostication than T analysis alone and could help in mCRC treatment management.

Hemoglobin level and XRCC1 polymorphisms to select patients with locally advanced rectal cancer candidate for neoadjuvant chemoradiotherapy with concurrent capecitabine and a platinum salt.

A platinum salt (oxaliplatin or cisplatin) is widely used to enhance chemoradation (CRT) response. The potential of cisplatin in neoadjuvant CRT for locally advanced rectal cancer (LARC) has not been fully investigated. Consecutive patients with histologically confirmed LARC were treated with standard pelvic radiotherapy and concurrent cisplatin plus capecitabine (CisCape CRT). Surgery and eight cycles of adjuvant FOLFOX4 were offered to all patients after CRT. Common biochemical variables and key germline genetic polymorphisms were analyzed as predictors of pathological complete response (pCR). Fifty-one patients were enrolled. pCR (regression AJCC grade 0) was documented in 7 patients (14%), nearly complete response (AJCC grade 1) in 10 pts. There was a strong association between disease-free survival and AJCC grade (p 0.0047). Grade 3-4 toxicities (mainly diarrhea) was observed in 41% of patients. Among all analyzed variables, baseline hemoglobin (Hb) was significantly associated with AJCC grade 0-1 response (p 0.027). As for the pharmacogenetic analysis, XRCC1 rs25487 polymorphism was significantly associated with AJCC grade 0-1, Odds Ratio 25.8, p 0.049. AJCC grade 0-1 response rate for patients with high Hb and/or XRCC1 rs25487 G/G genotype was as high as 57%. Baseline Hb and XRCC1 polymorphisms are valuable selection criteria for the CisCape CRT regimen, given its otherwise meaningful toxicity.

Expression and potential role of cellular retinol binding protein I in psoriasis.

Psoriasis is a diffuse chronic skin disorder characterized from accelerated epidermal turnover and inflammatory cell infiltrate. Retinoids influence keratinocyte proliferation and differentiation as well as inflammatory response. Cellular retinol binding protein (CRBPI) regulates intracellular vitamin A bioavailability and contributes to maintain skin homeostasis. The aim of present study was to investigate the expression of CRBPI and its role in the pathogenesis of skin psoriasis. Immunohistochemistry revealed more diffuse and increased CRBPI expression in all epidermal layers of human psoriatic lesions except in the stratum corneum. An imiquimod-induced psoriatic-like model documented the increase of skin lesional area and severity index score as well as of the severity of microscopic features as parakeratosis, papillomatosis and spongiosis in CRBPI-knockout compared to wild-type mice, associated to the increased keratinocyte CK17 and Ki-67 expression and the reduction of CK1, CRABPII and RXRα. Gene array of imiquimod-induced psoriatic skin documented the greater up-regulation of EGF/PDGF-related genes and down-regulation of EGR1 and pro-inflammatory IL-related genes in CRBPI-knockout compared to wild-type mice. Finally, CRBPI transfection in HaCaT cells increased AKT and NF-κB-related genes and proteins and down-regulated IL-2, IL-6 and IL-8 pro-inflammatory signalling. Although not recognized as a psoriatic susceptibility gene in our cohort of patients, the present data strongly supported the potential role of CRBPI to sustain keratinocyte proliferation and differentiation and to counteract pro-inflammatory genes expression in psoriatic lesions.

Loss of CRABP-II Characterizes Human Skin Poorly Differentiated Squamous Cell Carcinomas and Favors DMBA/TPA-Induced Carcinogenesis.

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-ß/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-ß/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-ß/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

Antioxidants and vascular health.

Oxygen free radicals and other reactive oxygen species (ROS) are common products of normal aerobic cellular metabolism, but high levels of ROS lead to oxidative stress and cellular damage. Increased production of ROS favors vascular dysfunction, inducing altered vascular permeability and inflammation, accompanied by the loss of vascular modulatory function, the imbalance between vasorelaxation and vasoconstriction, and the aberrant expression of inflammatory adhesion molecules. Inflammatory stimuli promote oxidative stress generated from the increased activity of mitochondrial nicotinamide adenine dinucleotide phosphate oxidase, particularly of the Nox4 isoform, with the consequent impairment of mitochondrial ß-oxidation. Vascular dysfunction due to the increase in Nox4 activity and ROS overproduction leads to the progression of cardiovascular diseases, diabetes, inflammatory bowel disease, and neurological disorders. Considerable research into the development of effective antioxidant therapies using natural derivatives or new synthetic molecules has been conducted. Antioxidants may prevent cellular damage by reducing ROS overproduction or interfering in reactions that involve ROS. Vitamin E and ascorbic acid are well known as natural antioxidants that counteract lipid peroxidative damage by scavenging oxygen-derived free radicals, thus restoring vascular function. Recently, preliminary studies on natural antioxidants such as goji berries, thymus, rosemary, green tea ginseng, and garlic have been conducted for their efficacy in preventing vascular damage. N-acetyl-cysteine and propionyl-L-carnitine are synthetic compounds that regulate ROS production by replacing endogenous antioxidants in both endothelial and smooth muscle cells. In this review, we consider the molecular mechanisms underlying the generation of oxidative stress-induced vascular dysfunction as well as the beneficial effects of antioxidant therapies.

Vitamin A, cancer treatment and prevention: the new role of cellular retinol binding proteins.

Retinol and vitamin A derivatives influence cell differentiation, proliferation, and apoptosis and play an important physiologic role in a wide range of biological processes. Retinol is obtained from foods of animal origin. Retinol derivatives are fundamental for vision, while retinoic acid is essential for skin and bone growth. Intracellular retinoid bioavailability is regulated by the presence of specific cytoplasmic retinol and retinoic acid binding proteins (CRBPs and CRABPs). CRBP-1, the most diffuse CRBP isoform, is a small 15 KDa cytosolic protein widely expressed and evolutionarily conserved in many tissues. CRBP-1 acts as chaperone and regulates the uptake, subsequent esterification, and bioavailability of retinol. CRBP-1 plays a major role in wound healing and arterial tissue remodelling processes. In the last years, the role of CRBP-1-related retinoid signalling during cancer progression became object of several studies. CRBP-1 downregulation associates with a more malignant phenotype in breast, ovarian, and nasopharyngeal cancers. Reexpression of CRBP-1 increased retinol sensitivity and reduced viability of ovarian cancer cells in vitro. Further studies are needed to explore new therapeutic strategies aimed at restoring CRBP-1-mediated intracellular retinol trafficking and the meaning of CRBP-1 expression in cancer patients' screening for a more personalized and efficacy retinoid therapy.

Brain natriuretic peptide modulates calcium homeostasis and epidermal growth factor receptor gene signalling in asthmatic airways smooth muscle cells.

The airway epithelium acts as a barrier and provides a critical interface between the body and the external environment. Brain natriuretic peptide (BNP) plays an important role in several bronchial functions, including relaxation. BNP relaxes airways by binding and activating natriuretic peptide receptor-A expressed from the airway epithelium. Although relaxation effect has been extensively investigated, less is known about BNP-regulated intracellular biomolecular pathways leading to bronchial relaxation. To this aim, we investigated BNP effects on gene signalling of airway smooth muscle cells (ASM) obtained from donors with asthma by using a RT(2) profiler™ PCR array. When compared with control, treatment for 2 h with supernatant from BNP-treated (1 µM) bronchial epithelial cells (BEAS-2B) induced in asthmatic ASM cells a rapid reduction of transcription of EGFR and genes involving in actin and calcium homeostasis, as those of Protein kinase C (PKC) and RhoA-ROCK gene pathways. Immunofluorescence and western blotting did not shown any difference comparing control and ASM cells treated with conditioned medium from BNP-treated BEAS-2B. This study provides evidence that the effect of BNP on relaxing bronchial in ASM cells is mediated from epithelium and associates to rapid changes of EGFR and calcium homeostasis-associated gene levels.

High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis.

PURPOSE: Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. EXPERIMENTAL DESIGN: CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. RESULTS: CRBP-1(High) expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1(High) status and dedifferentiation. CRBP-1-transfected (CRBP-1(+)) A549 grew more than CRBP-1(-) A549 cells. At >1µM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1(+) than in CRBP-1(-) A549 cells. CRBP-1(+) A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARß and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1(+) A549 cells. CONCLUSION: CRBP-1(High) expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1(High) expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients.

Antioxidant treatment prevents serum deprivation- and TNF-α-induced endothelial dysfunction through the inhibition of NADPH oxidase 4 and the restoration of ß-oxidation.

AIMS: Oxidative stress plays a pivotal role in the impaired endothelial function occurring in vascular diseases. Antioxidant strategies induce a clinical advantage in patients with endothelial dysfunction and atherosclerosis and protect from oxidative damage, but the underlying molecular mechanisms have been poorly evaluated. The aim of this study was to analyze the effects and mechanisms of action of antioxidant regimens on endothelial function. METHODS AND RESULTS: Antioxidant efficacy of N-acetylcysteine, ascorbic acid and propionyl-L-carnitine was evaluated in serum-deprived and TNF-α-stimulated human umbilical vein endothelial cells in vitro. Cell adhesion molecule (CAM) expression was evaluated by blot and real-time PCR, and inflammatory cytokine secretion was evaluated by ELISA; leukocyte adhesion and reactive oxygen species assays and NADPH oxidase 4 isoform (Nox4) expression analyses by blots were also performed. Antioxidant pretreatment restored serum-deprived and TNF-α-induced impaired mitochondrial ß-oxidation by reducing flavin adenine dinucleotide level and counteracting increased CAM and Nox4 expression, leukocyte adhesion and inflammatory cytokine secretion. Specific inhibition by plumbagin and siNox4 prevented TNF-α- and serum deprivation-induced detrimental effects, confirming that endothelial oxidative stress and inflammation were Nox4 dependent. CONCLUSIONS: Our findings documented Nox4 as a main actor in oxidative stress-induced endothelial dysfunction and further clarify the molecular basis of antioxidant treatment efficacy.

CRBP-1 expression in ovarian cancer: a potential therapeutic target.

BACKGROUND/AIM: Cellular retinol binding protein-1 regulates retinol bioavailability and contributes to cell differentiation maintenance, but its role in ovarian carcinogenesis remains uncertain. We investigated CRBP-1 expression in ovarian tumors and CRBP-1 signaling-regulated pathways. MATERIALS AND METHODS: We performed immunohistochemistry, methylation-specific PCR, gene copy number analysis in ovarian tumors and proliferation/apoptosis evaluation, gene array, blot and real-time PCR in CRBP-1-transfected A2780 ovarian cancer cells. RESULTS: CRBP-1 expression was reduced or absent in G2 and G3 ovarian carcinomas. CRBP-1 silencing in 60% of G2 and 66.7% of G3 carcinomas was due to CRBP-1 promoter methylation. A2780 CRBP-1-transfected cells showed increased retinol-induced apoptosis, retinoid-induced reduced clonogenicity and down-regulation of proliferation and transcription genes, including AKT1, AKT3, EGFR, FOS, JUN, STAT1 and STAT5A. CONCLUSION: CRBP-1 loss in G2/G3 ovarian carcinomas and increased apoptotic susceptibility to retinoids in CRBP-1-transfected-A2780 cells suggest CRBP-1 screening as a target to ensure efficacy of an adjuvant retinoid therapy.

High insulin-induced down-regulation of Erk-1/IGF-1R/FGFR-1 signaling is required for oxidative stress-mediated apoptosis of adipose-derived stem cells.

Homeostasis of adipose tissue requires highly coordinated response between circulating factors and cell population. Human adult adipose-derived stem cells (ASCs) display multiple differentiation properties and are sensitive to insulin stimulation. Insulin resistance and high level of circulating insulin characterize patients with type 2 diabetes and obesity. At physiological concentration, insulin promoted proliferation and survival of ASCs in vitro, whereas high insulin level induced their dose-dependent proliferative arrest and apoptosis. Insulin-induced apoptotic commitment depended on the down-regulation of Erk-1, insulin growth factor-1 receptor (IGF-1R), and fibroblast growth factor receptor-1 (FGFR-1)-mediated signaling. Specific inhibition of Erk-1/2, IGF-1R, and FGFR activity promoted ASC apoptosis but did not increase insulin effects, whereas EGFR and ErbB2 inhibition potentiated insulin-induced apoptosis. FGFRs and EGFR inhibition reduced ASC adipogenic differentiation, whereas Erk-1/2 and IGF-1R inhibition was ineffective. Insulin-induced apoptosis associated to reactive oxygen species (ROS) accumulation and inhibition of NADPH oxidase 4 (Nox4) activity prevented ASC apoptosis. Moreover, specific inhibition of Erk-1/2, IGF-1R, and FGFR-1 activity promoted ROS generation and this effect was not cumulative with that of insulin alone. Our data indicate that insulin concentration is a critical regulatory switch between proliferation and survival of ASCs. High insulin level-induced apoptotic machinery involves Nox4-generated oxidative stress and the down-regulation of a complex receptor signaling, partially distinct from that influencing adipogenic differentiation of ASCs.

Low-grade fibromyxoid sarcoma: an unusual cardiac location.

We report the unusual cardiac localization of a primary low-grade fibromyxoid sarcoma of the right ventricle in a 57-year-old woman. Histological examination revealed a prevalent myxoid appearance with whorling growth pattern of small or spindle cells with bland features alternating with rare more collagenous hypocellular areas with rare atypical cells. Genomic polymerase chain reaction of genomic DNA revealed the typical FUS/Creb3L2 fusion gene products typical of low-grade fibromyxoid sarcoma. The tumor was surgically removed and recurred after 7 years as high-grade pleomorphic sarcoma. The patient died 6 months after the clinical manifestation of recurrence. Low-grade fibromyxoid sarcoma of soft tissues is a rare, distinctive variant of fibrosarcoma-typically arising in deep soft tissue of lower extremities and trunk-that rarely metastasizes. Clinically, low-grade fibromyxoid sarcoma is characterized by a longer survival rate compared to other sarcomas, suggesting its consideration in the differential diagnosis of cardiac tumors with a myxoid appearance.

Platelet-rich plasma greatly potentiates insulin-induced adipogenic differentiation of human adipose-derived stem cells through a serine/threonine kinase Akt-dependent mechanism and promotes clinical fat graft maintenance.

The potential plasticity and therapeutic utility in tissue regeneration of human adipose-derived stem cells (ASCs) isolated from adult adipose tissue have recently been highlighted. The use of autologous platelet-rich plasma (PRP) represents an alternative strategy in regenerative medicine for the local release of multiple endogenous growth factors. Here we investigated the signaling pathways and effects of PRP and human recombinant insulin on proliferation and adipogenic differentiation of ASCs in vitro. PRP stimulated proliferation (EC(50) = 15.3 ± 1.3% vol/vol), whereas insulin's effect was the opposite (IC(50) = 3.0 ± 0.5 µM). Although PRP alone did not increase adipogenesis, in association with insulin it prevented ASC proliferative arrest, greatly enhanced intracytoplasmic lipid accumulation, strongly increased serine/threonine kinase Akt phosphorylation and mouse monoclonal anti-sterol regulatory element binding protein-1 accumulation, and downregulated Erk-1 activity; adipogenic effects were markedly prevented by the Akt inhibitor wortmannin. PRP with insulin synergistically upregulated fibroblast growth factor receptor (FGFR) and downregulated epidermal growth factor receptor (ErbB) expression; moreover, PRP in association prevented insulin-induced insulin-like growth factor-1 receptor and insulin receptor downregulation. The inhibition of FGFR-1, epidermal growth factor receptor (EGFR), and epidermal growth factor receptor-2 (ErbB2) activity reduced ASC proliferation, but only that of FGFR-1 reduced adipogenesis and Akt phosphorylation, whereas the ErbB2 inhibition effects were the opposite. However, EGFR activity was needed for ErbB2-mediated inhibition of ASC adipogenesis. Clinically, the injection of insulin further ameliorated patients' 1-year PRP-induced fat graft volume maintenance and contour restoring. Our results ascertain that PRP in association with insulin greatly potentiates adipogenesis in human ASCs through a FGFR-1 and ErbB2-regulated Akt mechanism. The ameliorated clinical fat graft maintenance suggests additional useful translational applications of combined PRP-insulin treatment in regenerative medicine.

Age-related increase of stem marker expression influences vascular smooth muscle cell properties.

OBJECTIVE: Aging represents a major risk factor for vascular disease development. With aging, changes of the biological properties of vascular smooth muscle cells (SMCs) are observed. Stem marker expression characterizes SMCs during developmental growth and atherosclerosis, but the contribution of SMCs with stem features to the age-related arterial remodeling remains largely unknown. METHODS AND RESULTS: Immunostaining revealed rare vascular growth factor receptor-1(+) (flt-1(+)) and c-kit(+) cells in tunica media of grossly normal human young (17-30 years old) large arteries and 2-month old rat aorta, whereas CD133(+) cells were absent. In large arteries of human aged donors (64-77 years), flt-1(+) and c-kit(+) cell number increased in the intimal thickening and tunica media. Double immunofluorescence revealed that 30.6 ± 3% of flt-1(+) intimal cells co-expressed α-smooth muscle actin. Immunostaining, blots and RT-PCR documented the increased expression of flt-1 and c-kit in 20-24-month old rat aortic media. In vitro, old rat aortic SMCs proliferated and migrated more with greater flt-1, c-kit, NF-κB, VCAM-1, IAP-1 and MCP-1 levels and less α-smooth muscle actin and myosin compared to young SMCs. Old SMCs were also more susceptible to all-trans retinoic and NF-κB inhibition-induced apoptosis compared to young SMCs. Anti-flt-1 blocking antibody reduced migration and placental growth factor-induced but not serum and PDGF-BB-stimulated proliferation of old SMCs. CONCLUSIONS: The increase of flt-1(+) and c-kit(+) SMCs characterizes large arteries of aged donors; the blocking of flt-1 signaling influences the behavior of old SMCs, suggesting that the accumulation of SMCs with a stem phenotype contributes to the age-dependent adverse arterial remodeling.

Inverse correlation between VEGF and soluble VEGF receptor 2 in POEMS with AIDP responsive to intravenous immunoglobulin.

POEMS (polyneuropathy, organomegaly, endocrinopathy, M-band, and skin changes) syndrome is characterized by chronic progressive polyneuropathy and plasma-cell dyscrasia. A major diagnostic criterion of POEMS is elevation of circulating vascular endothelial growth factor (VEGF), which is believed to play a pathogenic role in this disease. We report a case of POEMS that presented as relapsing acute inflammatory demyelinating polyneuropathy, in which complete remission after intravenous immunoglobulin (IVIg) treatment was unexpectedly observed. At clinical nadir, the VEGF level was 30-fold higher, and the soluble form of VEGF receptor 2 (sVEGFR2), which acts as a decoy for VEGF, was 2.7-fold lower than normal. These changes combined might contribute to the pathogenesis of POEMS, inducing vascular permeability and tissue edema. At 9-month follow-up, during clinical remission, VEGF and sVEGFR2 were near normal values. sVEGFR2 reduction is a new finding in POEMS. IVIg treatment may benefit POEMS patients with acute neuropathy by downgrading VEGF release induced by inflammatory cytokines.