Heparin Modulates the Mitogenic Activity of Fibroblast Growth Factor by Inducing Dimerization of its Receptor. A 3D View by Using NMR

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin‐binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin‐binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin‐binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF‐driven mitogenesis.     

Pivotal Role of Corneal Fibroblasts in Progression to Corneal Ulcer in Bacterial Keratitis

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.     

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.     

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.     

蓝莓花色苷对鸡胚成纤维细胞系生长的影响

以花色苷含量为21.34%的蓝莓花色苷粉末为原料,采用MTT法、显微镜观察法,同时利用细胞计数仪测定细胞活率,3种方法共同检测蓝莓花色苷作用于鸡胚成纤维细胞系的细胞毒性,以完成细胞试验模型的建立。结果表明:50~100μg/mL的蓝莓花色苷可以促进鸡胚成纤维细胞生长,高于100μg/mL的蓝莓花色苷,对细胞生长产生抑制作用。该结果可为进一步拓展花色苷功能性研究,以及以鸡胚成纤维细胞为模型的生物功能性研究提供理论依据。     

Pathogenic Mechanisms of Hypertrophic Cardiomyopathy beyond Sarcomere Dysfunction

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, affecting 1 in 500 people in the general population. Although characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, and cardiac fibrosis, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset, and complications. While HCM is generally accepted as a disease of the sarcomere, variable penetrance in families with identical genetic mutations challenges the monogenic origin of HCM and instead implies a multifactorial cause. Furthermore, large-scale genome sequencing studies revealed that many genes previously reported as causative of HCM in fact have little or no evidence of disease association. These findings thus call for a re-evaluation of the sarcomere-centered view of HCM pathogenesis. Here, we summarize our current understanding of sarcomere-independent mechanisms of cardiomyocyte hypertrophy, highlight the role of extracellular signals in cardiac fibrosis, and propose an alternative but integrated model of HCM pathogenesis.     

The Antimicrobial Peptides Human β-Defensins Induce the Secretion of Angiogenin in Human Dermal Fibroblasts

The skin produces a plethora of antimicrobial peptides that not only show antimicrobial activities against pathogens but also exhibit various immunomodulatory functions. Human β-defensins (hBDs) are the most well-characterized skin-derived antimicrobial peptides and contribute to diverse biological processes, including cytokine production and the migration, proliferation, and differentiation of host cells. Additionally, hBD-3 was recently reported to promote wound healing and angiogenesis, by inducing the expression of various angiogenic factors and the migration and proliferation of fibroblasts. Angiogenin is one of the most potent angiogenic factors; however, the effects of hBDs on angiogenin production in fibroblasts remain unclear. Here, we investigated the effects of hBDs on the secretion of angiogenin by human dermal fibroblasts. Both in vitro and ex vivo studies demonstrated that hBD-1, hBD-2, hBD-3, and hBD-4 dose-dependently increased angiogenin production by fibroblasts. hBD-mediated angiogenin secretion involved the epidermal growth factor receptor (EGFR), Src family kinase, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-kappa B (NF-κB) pathways, as evidenced by the inhibitory effects of specific inhibitors for these pathways. Indeed, we confirmed that hBDs induced the activation of the EGFR, Src, JNK, p38, and NF-κB pathways. This study identified a novel role of hBDs in angiogenesis, through the production of angiogenin, in addition to their antimicrobial activities and other immunomodulatory properties.     

A New Hypothetical Concept in Metabolic Understanding of Cardiac Fibrosis: Glycolysis Combined with TGF-β and KLF5 Signaling

The accumulation of fibrosis in cardiac tissues is one of the leading causes of heart failure. The principal cellular effectors in cardiac fibrosis are activated fibroblasts and myofibroblasts, which serve as the primary source of matrix proteins. TGF-β signaling pathways play a prominent role in cardiac fibrosis. The control of TGF-β by KLF5 in cardiac fibrosis has been demonstrated for modulating cardiovascular remodeling. Since the expression of KLF5 is reduced, the accumulation of fibrosis diminishes. Because the molecular mechanism of fibrosis is still being explored, there are currently few options for effectively reducing or reversing it. Studying metabolic alterations is considered an essential process that supports the explanation of fibrosis in a variety of organs and especially the glycolysis alteration in the heart. However, the interplay among the main factors involved in fibrosis pathogenesis, namely TGF-β, KLF5, and the metabolic process in glycolysis, is still indistinct. In this review, we explain what we know about cardiac fibroblasts and how they could help with heart repair. Moreover, we hypothesize and summarize the knowledge trend on the molecular mechanism of TGF-β, KLF5, the role of the glycolysis pathway in fibrosis, and present the future therapy of cardiac fibrosis. These studies may target therapies that could become important strategies for fibrosis reduction in the future.     

A Novel FGFR1 Missense Mutation in a Portuguese Family with Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive endocrine disorder characterized by complete or partial failure of pubertal development and infertility due to deficiency of the gonadotropin-releasing hormone (GnRH). CHH has a significant clinical heterogeneity and can be caused by mutations in over 30 genes. The aim of this study was to investigate the genetic defect in two siblings with CHH. A woman with CHH associated with anosmia and her brother with normosmic CHH were investigated by whole exome sequencing. The genetic studies revealed a novel heterozygous missense mutation in the Fibroblast Growth Factor Receptor 1 (FGFR1) gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_023110.3","term_id":"1732262558"}}NM_023110.3: c.242T>C, p.Ile81Thr) in the affected siblings and in their unaffected father. The mutation affected a conserved amino acid within the first Ig-like domain (D1) of the protein, was predicted to be pathogenic by structure and sequence-based prediction methods, and was absent in ethnically matched controls. These were consistent with a critical role for the identified missense mutation in the activity of the FGFR1 protein. In conclusion, our identification of a novel missense mutation of the FGFR1 gene associated with a variable expression and incomplete penetrance of CHH extends the known mutational spectrum of this gene and may contribute to the understanding of the pathogenesis of CHH.     

用农杆菌真空渗透法在烟草中瞬时表达人酸性成纤维细胞生长因子

利用农杆菌真空渗透法在烟草中瞬时表达了人酸性成纤维细胞生长因子(haFGF)基因,并对影响表达量的几个因素进行了研究.结果表明,在真空渗透后4天,外源基因获得最高水平的表达,烟草Nicotiana benthamiana(N.b)的表达量明显低于烟草N.H(转基因沉默抑制子Hc-pro的N.b植株).pM390-haFGF、pM390-haFGF/KDEL、pM390-SP/haFGF/KDEL三种表达框架被用来表达人酸性成纤维细胞生长因子,结果表明框架pM390-haFGF/KDEL的表达效果最好,受伤叶片和Valentine的培养方法要比完整叶片和Kapila的培养方法的表达量高.