Differential effects of cyclic stretch on bFGF‐ and VEGF‐induced sprouting angiogenesis

How mechanical factors affect angiogenesis and how they and chemical angiogenic factors work in concert remain not yet well‐understood. This study investigated the interactive effects of cyclic uniaxial stretch and two potent proangiogenic molecules [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)] on angiogenesis using a stretchable three‐dimensional (3‐D) cell culture model. Endothelial cells seeded atop a 3‐D collagen gel underwent sprouting angiogenesis while being subjected to either 10 or 20% cyclic uniaxial stretch at a frequency of either 1/12 or 1 Hz, in conjunction with an elevated concentration of bFGF or VEGF. Without the presence of additional growth factors, 10 and 20% stretch at 1 Hz induced angiogenesis and the perpendicular alignment of new sprouts, and both inductive effects were abolished by cytochalasin D (an actin polymerization inhibitor). While “10% stretch at 1 Hz,” “20% stretch at 1 Hz,” bFGF, and VEGF were strong angiogenesis stimulants individually, only the combination of “20% stretch at 1 Hz” and bFGF had an additive effect on inducing new sprouts. Interestingly, the combination of “20% stretch at a lower frequency (1/12 Hz)” and bFGF decreased sprouting angiogenesis, even though the level of perpendicular alignment of new sprouts was the same for both stretch frequencies. Taken together, these results demonstrate that both stretch frequency and magnitude, along with interactions with various growth factors, are essential in mediating formation of endothelial sprouts and vascular patterning. Furthermore, work in this area is warranted to elucidate synergistic or competitive signaling mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:879–888, 2014     

Serum concentrations of insulin‐like growth factor‐i and insulin‐like growth factor binding protein‐2 and ‐3 in eight hoofstock species

The somatotropic axis, which includes growth hormone, insulin‐like growth factor (IGF)‐I, and IGF binding proteins (IGFBP), is involved in the regulation of growth and metabolism. Measures of the somatotropic axis can be predictive of nutritional status and growth rate that can be utilized to identify nutritional status of individual animals. Before the somatotropic axis can be a predictive tool, concentrations of hormones of the somatotropic axis need to be established in healthy individuals. To begin to establish these data, we quantified IGF‐I, IGFBP‐2, and IGFBP‐3 in males and females of eight threatened hoofstock species at various ages. Opportunistic blood samples were collected from Bos javanicus (Java banteng), Tragelaphus eurycerus isaaci (bongo), Gazella dama ruficollis (addra gazelle), Taurotragus derbianus gigas (giant eland), Kobus megaceros (Nile lechwe), Hippotragus equines cottoni (roan antelope), Ceratotherium simum simum (white rhinoceros), and Elephas maximus (Asian elephant). Serum IGF‐I and IGFBPs were determined by radioimmunoassay and ligand blot, respectively. Generally, IGF‐I and IGFBP‐3 were greater in males, and IGFBP‐2 was greater in females. In banteng (P = 0.08) and male Nile lechwe (P<0.05), IGF‐I increased with age, but decreased in rhinoceros (P = 0.07) and female Nile lechwe (P<0.05). In banteng, IGFBP‐3 was greater (P<0.01) in males. In elephants (P<0.05) and antelope (P = 0.08), IGFBP‐2 were greater in females. Determination of concentrations of hormones in the somatotropic axis in healthy animals makes it possible to develop models that can identify the nutritional status of these threatened hoofstock species. Zoo Biol 30:275–284, 2011. © 2010 Wiley‐Liss, Inc.     

Cross‐talk between the VEGF‐A and HGF signalling pathways in endothelial cells

Background information. Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre‐existing vascular bed. VEGF‐A (vascular endothelial growth factor‐A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF‐A‐driven angiogenesis, although the signalling mechanisms underlying this co‐operation are not completely understood. Results. We analysed the effects of the combination of VEGF‐A and HGF on the activation of VEGFR‐2 (VEGF receptor‐2) and c‐met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR‐2 and c‐met do not physically associate and do not transphosphorylate each other, suggesting that co‐operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF‐A₁₆₅ and HGF stimulate a similar set of MAPKs (mitogen‐activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF‐A₁₆₅ and HGF. Moreover, the combination of VEGF‐A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF‐A₁₆₅ and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF‐A₁₆₅ and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular‐like patterns in vitro in a Rho‐ or Rac‐dependent manner. Conclusions. Under angiogenic conditions, combining VEGF‐A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.     

Sympathetic neurons synthesize and secrete pro‐nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF‐immunoreactive proteins synthesized by cultured NGF‐dependent and ‐independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro‐NGF protein. These findings suggest that a potential NGF‐sympathetic neuron autocrine loop may exist in this prototypic target‐dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival. © 2003 Wiley Periodicals, Inc. J Neurobiol 38–53, 2003     

miR‐149 controls non‐alcoholic fatty liver by targeting FGF‐21

Non‐alcoholic fatty liver disease (NAFLD), a lipid metabolism disorder characterized by the accumulation of intrahepatic fat, has emerged as a global public health problem. However, its underlying molecular mechanism remains unclear. We previously have found that miR‐149 was elevated in NAFLD induced by high‐fat diet mice model, whereas decreased by a 16‐week running programme. Here, we reported that miR‐149 was increased in HepG2 cells treated with long‐chain fatty acid (FFA). In addition, miR‐149 was able to promote lipogenesis in HepG2 cells in the absence of FFA treatment. Moreover, inhibition of miR‐149 was capable of inhibiting lipogenesis in HepG2 cells in the presence of FFA treatment. Meanwhile, fibroblast growth factor‐21 (FGF‐21) was identified as a target gene of miR‐149, which was demonstrated by the fact that miR‐149 could negatively regulate the protein expression level of FGF‐21, and FGF‐21 was also responsible for the effect of miR‐149 inhibitor in decreasing lipogenesis in HepG2 cells in the presence of FFA treatment. These data implicate that miR‐149 might be a novel therapeutic target for NAFLD.     

Fibroblast growth factor‐2/platelet‐derived growth factor enhances atherosclerotic plaque stability

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor‐A (VEGF)‐A, fibroblast growth factor (FGF)‐2, platelet‐derived growth factor (PDGF)‐BB and FGF‐2 + PDGF‐BB. Lentivirus was percutaneously injected into the media‐adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque‐rupture rate, plaque‐vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF‐2/PDGF‐BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF‐A‐ and FGF‐2‐overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF‐2/PDGF‐BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF‐2/PDGF‐BB induced epsin‐2 expression and enhanced the VEGF receptor‐2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF‐2 and PDGF‐BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

LncRNA TNK2‐AS1 regulated ox‐LDL‐stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR‐150‐5p

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.     

Differential Roles of Grb2 and AP‐2 in p38 MAPK‐ and EGF‐Induced EGFR Internalization

The epidermal growth factor receptor ( EGFR ) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand‐induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF) ‐induced EGFR internalization also required Grb 2 , p38 MAPK ‐induced internalization did not. Interestingly , AP ‐2 knock down blocked p38 MAPK ‐induced EGFR internalization, but only mildly affected EGF ‐induced internalization. In line with this, simultaneously mutating two AP ‐2 interaction sites in EGFR affected p38 MAPK ‐induced internalization much more than EGF ‐induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.     

Anti‐EGFR antibody‐drug conjugate for triple‐negative breast cancer therapy

Triple‐negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody‐drug conjugate (ADC) to target EGFR⁺ TNBC and deliver high‐potency drug. First, we constructed an ADC by conjugating anti‐EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo‐SMCC. Second, we confirmed the TNBC‐targeting specificity of anti‐EGFR ADC by evaluating its surface binding and internalization in MDA‐MB‐468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live‐cell and live‐animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC‐targeting. Finally, both in vitro toxicity assay and in vivo anti‐cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti‐EGFR ADC has a great potential to against TNBC.     

Expressions of Leptin and Insulin‐Like Growth Factor‐I Are Highly Correlated and Region‐Specific in Adipose Tissue of Growing Rats

Objective: Anatomically distinct adipose tissue regions differ in their predominant modality of growth (i.e., cellular hypertrophy vs. hyperplasia). We examined site‐specific patterns of expression of two genes whose products, leptin and insulin‐like growth factor‐I (IGF‐I), could be involved in mediating differential growth and metabolism of white adipose tissue. We also related these patterns of expression to measures of adipose depot cellularity. Research Methods and Procedures: Male Wistar rats were fed ad libitum and studied from ages 7 weeks to ～12 months. Terminal measures of body weights; weights, composition, and cellularity of four white adipose depots; circulating leptin and IGF‐I; and adipose depot‐specific expression levels of leptin and IGF‐I were measured in subsets of rats at 7, 12, 22, 42, and 46 weeks of age. Results: Both leptin and IGF‐I mRNAs are quantitatively expressed in a depot‐specific manner, in the following order: retroperitoneal ? epididymal > mesenteric > subcutaneous inguinal. Furthermore, there is a marked correlation between the expressions of these hormones in the various regions of adipose tissue of rats during the first year of life. The mechanisms that underlie the parallel expressions of leptin and IGF‐I appear to be related to fat‐cell volume. Discussion: Because both leptin and IGF‐I have been implicated in the regulation of energy homeostasis and are both expressed in adipose tissue, the depot‐specific linkage between the two genes suggests interaction at the autocrine level. This interaction may have an important role in determining functional properties particular to individual adipose depots.     

Retinitis Pigmentosa: over‐expression of anti‐ageing protein Klotho in degenerating photoreceptors

Retinitis Pigmentosa involves a hereditary degeneration of photoreceptors by as yet unresolved mechanisms. The secretable protein α‐Klotho has a function related to ageing processes, and α‐Klotho‐deficient mice have reduced lifespan and declining functions in several tissues. Here, we studied Klotho in connection with inherited photoreceptor degeneration. Increased nuclear immunostaining for α‐Klotho protein was seen in degenerating photoreceptors in four different Retinitis Pigmentosa models (rd1, rd2 mice; P23H, S334ter rhodopsin mutant rats). Correspondingly, in rd1 retina α‐Klotho mRNA expression was significantly up‐regulated. Moreover, immunostaining for another Klotho family protein, β‐Klotho, also co‐localized with degenerating rd1 photoreceptors. The rd1 retina displayed reduced levels of fibroblast growth factor 15, a member of the fibroblast growth factor subfamily for which Klotho acts as a co‐receptor. Exogenous α‐Klotho protein added to retinal explant cultures did not affect cell death in rd1 retinae, but caused a severe layer disordering in wild‐type retinae. Our study suggests Klotho as a novel player in the retina, with a clear connection to photoreceptor cell death as well as with an influence on retinal organization.     

TGF‐β1 monoclonal antibody: Assessment of embryo‐fetal toxicity in rats and rabbits

A humanized monoclonal antibody targeting transforming growth factor β1 (TGF‐β1 mab) has been used in development for the treatment of chronic kidney disease. Embryo‐fetal development studies were conducted in rats and rabbits using 30 and 25 animals per group, respectively. The TGF‐β1 mab was administered subcutaneously to rats at 0, 2, or 50 mg/kg/dose on gestation days (GDs) 6, 10, and 14 and intravenously to rabbits at 0 or 3 mg/kg/dose on GDs 7, 12 to 19, and at 30 mg/kg/dose on GDs 7, 12, 14, 16, and 18. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated. There was no indication of maternal or embryo‐fetal toxicity in the rat. Effects in the rabbit were limited to the fetus where the 30 mg/kg TGF‐β1 mab dose produced a slight decrease in fetal weight and an increase in the incidence of retrocaval ureter and an absent and/or malpositioned kidney/ureter in two fetuses. In conclusion, TGF‐β1 mab produced no adverse maternal or embryo‐fetal findings in rats when administered ≤50 mg/kg on GDs 6, 10, and 14. TGF‐β1 mab did not demonstrate maternal toxicity or embryo‐fetal lethality at doses as high as 30 mg/kg when administered on GDs 7, 12, 14, 16, and 18 in rabbits. Fetal growth and morphology were affected only at 30 mg/kg; thus, the no observed adverse effect level was 3 mg/kg in rabbits. The margin of safety for both rats and rabbits was ≥37‐fold the clinical exposure level.     

FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

Fibroblast growth factor 21 (FGF21) acts as an anti‐atherosclerotic agent. However, the specific mechanisms governing this regulatory activity are unclear. Autophagy is a highly conserved cell stress response which regulates atherosclerosis (AS) by reducing lipid droplet degradation in foam cells. We sought to assess whether FGF21 could inhibit AS by regulating cholesterol metabolism in foam cells via autophagy and to elucidate the underlying molecular mechanisms. In this study, ApoE^?/? mice were fed a high‐fat diet (HFD) with or without FGF21 and FGF21 + 3‐Methyladenine (3MA) for 12 weeks. Our results showed that FGF21 inhibited AS in HFD‐fed ApoE^?/? mice, which was reversed by 3MA treatment. Moreover, FGF21 increased plaque RACK1 and autophagy‐related protein (LC3 and beclin‐1) expression in ApoE^?/? mice, thus preventing AS. However, these proteins were inhibited by LV‐RACK1 shRNA injection. Foam cell development is a crucial determinant of AS, and cholesterol efflux from foam cells represents an important defensive measure of AS. In this study, foam cells were treated with FGF21 for 24 hours after a pre‐treatment with 3MA, ATG5 siRNA or RACK1 siRNA. Our results indicated that FGF21‐induced autophagy promoted cholesterol efflux to reduce cholesterol accumulation in foam cells by up‐regulating RACK1 expression. Interestingly, immunoprecipitation results showed that RACK1 was able to activate AMPK and interact with ATG5. Taken together, our results indicated that FGF21 induces autophagy to promote cholesterol efflux and reduce cholesterol accumulation in foam cells through RACK1‐mediated AMPK activation and ATG5 interaction. These results provided new insights into the molecular mechanisms of FGF21 in the treatment of AS.