Single-cell analysis of human dermal fibroblasts isolated from a single male donor over 35 years.

The aim of this study is to examine the effects of ageing on dermal fibroblast heterogeneity based on samples obtained from the same donor. We used a dermal fibroblast lineage (named ASF-4 cell lines) isolated from the inner side of the upper arm of a healthy male donor over a 35-year period, beginning at 36 years of age. Because clonal analysis of ASF-4 cell lines demonstrated a donor age-dependent loss of proliferative capacity and acquisition of senescent traits at the single-cell level, cultured cells frozen at passage 10 at ages 36 and 72 years were subjected to single-cell RNA sequencing. Transcriptome analysis revealed an increase in senescent fibroblasts and downregulation of genes associated with extracellular matrix remodelling with ageing. In addition, two putative differentiation pathways, with one endpoint consisting of senescent fibroblasts and the other without, were speculated using a pseudo-time analysis. Knockdown of the characteristic gene of the non-senescent fibroblast cluster endpoint, EFEMP2, accelerated cellular senescence. This was also confirmed in two other normal human dermal fibroblast cell lines. The detection of a common cellular senescence-related gene from single-donor analysis is notable. This study provides new insights into the behaviour of dermal fibroblasts during skin ageing.

Brazilian Propolis Suppresses Angiogenesis by Inducing Apoptosis in Tube-Forming Endothelial Cells through Inactivation of Survival Signal ERK1/2.

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.

Antiangiogenic effects of indole-3-carbinol and 3,3'-diindolylmethane are associated with their differential regulation of ERK1/2 and Akt in tube-forming HUVEC.

We previously reported that indole-3-carbinol (I3C), found in cruciferous vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying molecular mechanisms still remain unclear. Antiangiogenic effects of its major metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In this study, we investigated the effects of these indoles on angiogenesis and tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by affecting angiogenic signal transduction in human umbilical vein endothelial cells (HUVEC). We found that I3C and DIM at 25 micromol/L significantly inhibited tube formation and only DIM induced a significant increase in apoptosis in tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At the molecular level, I3C and DIM markedly inactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was significantly greater than that of I3C. DIM treatment also resulted in activation of the caspase pathway and inactivation of Akt, whereas I3C did not affect them. These results indicate that I3C and DIM had a differential potential in the regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt was enough to inhibit tube formation and induce caspase-dependent apoptosis in tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is responsible for its stronger antiangiogenic effects than those of I3C.

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells.

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.

MOV10 as a novel telomerase-associated protein.

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.

Involvement of DNA fragmentation of enterocytes in mucosal injury to a mouse jejunum incubated in ussing chambers.

A mouse jejunum, when incubated in vitro in Ussing chambers, was found to exhibit morphological deterioration of the villi with denudation of the epithelia (J Nutr Sci Vitaminol, 51: 406, 2005). Our study examined the involvement of apoptosis in an intestinal injury model by a DNA ladder assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Electrophoresis of mucosal DNA revealed ladders, indicating the occurrence of DNA fragmentation. Cells with TUNEL-positive nuclei were detected among the villus epithelial cells (enterocytes), whereas they are rarely seen among crypt epithelial cells. These features were evident within 1 h after the start of incubation. Apoptotic death of the enterocytes was thus involved in the destruction of villi when incubated in Ussing chambers.

Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice.

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Hypoxia induces apoptosis of HUVECs in an in vitro capillary model by activating proapoptotic signal p38 through suppression of ERK1/2.

We recently reported that hypoxia induces chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, to tube-forming HUVECs in an in vitro blood vessel model by activating p38 MAPK. In this report, we further examined what role p38 plays and how it is activated during hypoxia-induced apoptosis. First, in order to confirm that p38 can indeed induce apoptosis, the cells were treated with anisomycin, a p38 activator, during normoxia. The activator treatment induced apoptosis and activation of p38 and caspase-3 in a very short time, which indicated that p38 activation alone was sufficient to trigger apoptosis in tube-forming HUVECs. We then observed hypoxia-induced changes in intracellular signals, ERK1/2 and Akt. ERK1/2 inactivation was shown to occur prior to p38 activation and caspase-3 cleavage during hypoxia. On the other hand, anisomycin had no inhibitory effect on ERK1/2 activation during normoxia. It was also shown that the amount of Akt protein slightly decreased by either hypoxia or anisomycin treatment. We then investigated how these two survival signals, ERK1/2 and Akt, are involved in p38 activation by using MEK inhibitor U0126 and PI3K inhibitor LY294002. When tube-forming HUVECs were treated with U0126 or LY294002 during normoxia, the two inhibitors were able to induce apoptosis and activation of p38 and caspase-3 in a relatively short time. U0126 was able to inhibit ERK1/2 activation, but had almost no effect on Akt activation. In contrast, LY294002 was able to inhibit Akt activation, but had very little effect on ERK1/2 activation. These results indicate that ERK1/2 inactivation, rather than Akt decrease, is responsible for hypoxia-induced p38 activation. Taken together, our results strongly suggest that hypoxia-induced apoptosis is regulated through signal transduction in which inactivation of ERK1/2 leads to activation of p38, which then triggers caspase cascade as an execution mechanism of apoptosis.

Increased levels of a particular phosphatidylcholine species in senescent human dermal fibroblasts in vitro.

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.

Propolis suppresses tumor angiogenesis by inducing apoptosis in tube-forming endothelial cells.

We have reported that propolis suppresses tumor-induced angiogenesis in vivo and in vitro, but antiangiogenic mechanism of propolis at cellular level remains unclear. In this study, we observed that propolis not only inhibited tube formation but also induced apoptosis of endothelial cells. These results suggest that propolis exerts its antiangiogenic effects at least in part through induction of apoptosis.

Indole-3-carbinol suppresses tumor-induced angiogenesis by inhibiting tube formation and inducing apoptosis.

Indole-3-carbinol (I3C) has been reported to exert anticancer activity in vivo. However, its anticancer mechanism has not been fully elucidated. In this study, we demonstrate that I3C suppressed tumor-induced angiogenesis and tube formation of endothelial cells. I3C also induced apoptosis in endothelial cells by activating the caspase cascade. We propose that I3C exerts its anticancer effect through the induction of endothelial apoptosis.

Suppression of tumor-induced angiogenesis by Brazilian propolis: major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation.

Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis.

Tea catechins inhibit angiogenesis in vitro, measured by human endothelial cell growth, migration and tube formation, through inhibition of VEGF receptor binding.

We have investigated whether tea catechins (EC, ECg, EGC, EGCg) have any inhibitory effects on angiogenesis and which step they affect during the process. The effects of catechins were tested on in vitro models of angiogenesis, namely, growth, migration and tube formation of human umbilical vein endothelial cells. All four catechins inhibited angiogenesis in vitro in the three different bioassays with concentrations ranging from 1.56 to 100 microM. Among the four catechins tested, epigallocatechin gallate (EGCg) was the most effective in inhibiting angiogenesis in all three assays. When these four catechins were tested on VEGF binding assay, only EGCg inhibited the binding of VEGF, a major angiogenesis inducing factor, to endothelial cells in a concentration dependent manner. These results indicate that while all four tea catechins inhibit the process of angiogenesis, EGCg alone can reduce the binding of VEGF to its receptors and thus affects the downstream signaling.

Degradation of nonylphenol polyethoxylates by ultraviolet B irradiation and effects of their products on mammalian cultured cells.

Nonylphenol polyethoxylates (NPEOs) are widely used as non-ionic surfactants and their biodegradation products such as 4-n-nonylphenol are stable and have been demonstrated to be cytotoxic. In the aquatic environment, these compounds are usually exposed to sunlight, and while the correlation between the biodegradation of NPEOs and changes in cytotoxicity has been reported, the relationship between the photodegradation of NPEOs and cytotoxicity has not. In this study, we investigated the degradation of NPEO by ultraviolet (UV) irradiation, especially UVB irradiation, and the effects on mammalian cell lines. NPEO with a smaller number of ethylene oxide (EO) units showed greater cytotoxicity. Although NPEO (10) completely inhibited the proliferation of the cells, NPEO (70) showed no toxicity. UVB irradiation significantly induced a shortening of the side chain, which was due to the production of ROS. The EO side chain of NPEO (10), was gradually degradated, but that of NPEO (70) was degradated near the benzene ring. Furthermore, the degradation of the benzene ring was more effective in NPEO (70) than NPEO (10). The toxicity of NPEO (10) in cultured cells decreased following UVB irradiation, whereas that of NPEO (70) was induced after UVB irradiation at 500 J/cm2 and disappeared at 1000 J/cm2. This might be due to the production of NPEO with a short side chain and 4-n-nonylphenol by the degradation of EO and due to the degradation of the benzene ring at higher doses of UVB irradiation. This study shows the significance of UV exposure to the degradation of alkylphenol polyethoxylates in the environment.

Effect of quercetin conjugates on vascular permeability and expression of adhesion molecules.

Quercetin and its glucosides exist in plant foods and are recovered in human plasma as glucuronide and sulfate conjugates. Quercetin and its conjugates show antioxidant activity in model experiments. It remains obscure, however, whether these conjugates retain these biological functions in vivo. We investigated the interaction of quercetin conjugates with lipid bilayers using liposome systems. Less quercetin conjugate was incorporated into liposomes than quercetin aglycone. We also studied the vascular permeability of quercetin-3-glucuronide using cell culture inserts. Incubation of human aortic endothelial cells (HAECs) with IL-1alpha resulted in increased permeability of quercetin-3-glucuronide. Furthermore, quercetin-3-glucuronide showed no suppressive effect on TNF-alpha-induced cell expression of intercellular adhesion molecule-1 (ICAM-1) on HAECs. These observations suggest that circulating conjugates of quercetin pass through the endothelium to reach vascular smooth muscle cells and exert their biological effects in the blood vessels during inflammation followed by deconjugation of the conjugates.

Expression and release of progalanin in fibroblasts.

Galanin is a neuropeptide expressed in the central and peripheral nervous systems. Galanin is known to be biosynthesized in neural and endocrine cells, but little evidence exists for its synthesis in other cells. In this study, we explored galanin-releasing nonneural cells using radioimmunoassay, finding that some fibroblasts produced and released the galanin-like immunoreactive component (galanin-LI). The molecular weight of the galanin-LI obtained from the fibroblasts, as measured by gel filtration chromatography and Western blotting, was 14 kDa and suggested that the compound was progalanin. Peptide mass fingerprinting analysis identified the large form of galanin-LI as progalanin without its signal sequence. In addition, galanin-LI was located in the Golgi bodies and vesicle-like structures of the fibroblasts. Furthermore, the addition of brefeldin A, an inhibitor of transport from the ER, decreased the release of galanin-LI. In this study, we showed that the fibroblast, a nonneural and nonendocrine cell type, produced and released a galanin precursor, progalanin, without processing via Golgi bodies or secretory vesicles.

FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.

Calcineurin inhibitors such as cyclosporin A (CsA) and FK506 have been used in solid organ and hematopoietic stem cell transplantations to suppress immune function. However, these immunosuppresants are associated with severe endothelial dysfunction. We investigated whether CsA and FK506 induce endothelial dysfunction using a three-dimensional culture blood vessel model, in which human umbilical vein endothelial cells form and maintain capillary-like tube and lumen structures. We found that FK506, but not CsA, induced breakdown of the tube structures and endothelial cell death. FK506 inhibited calcineurin activity, but FK506-induced tube breakdown and cell death was not suppressed by RNA interference targeting calcineurin Aα. FK506 also induced caspase activation, but caspase inhibition by zVAD(OMe)-fmk failed to suppress FK506-induced tube breakdown and cell death. FK506 induced attenuation of Akt and extracellular-regulated kinase 1/2 (ERK1/2). Furthermore, Akt inhibition by LY294002 or ERK1/2 inhibition by PD98059 induced tube breakdown and cell death. Present results suggest that FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway.

Resveratrol derivative-rich melinjo (Gnetum gnemon L.) seed extract suppresses multiple angiogenesis-related endothelial cell functions and tumor angiogenesis.

Angiogenesis is a promising target for cancer prevention and treatment. This study aimed to determine the antiangiogenic effects of melinjo (Gnetum gnemon L.) seed extract and its resveratrol derivative components, such as gnetin C (GC), gnetin L (GL), gnemonoside A (GMA), gnemonoside C (GMC), and gnemonoside D (GMD). An ethanol extract of melinjo seeds (EEMS) and the two gnetins markedly inhibited the proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor and basic fibroblast growth factor. The inhibitory effects of GC and GL were much stronger than those of resveratrol. GMC and GMD inhibited only proliferation, whereas GMA had almost no effect on the two endothelial cell functions. The EEMS and GC also reduced the cell viability of tube-forming HUVEC, with accompanying ERK1/2 inactivation, and suppressed the migration of HUVEC. Furthermore, dietary intake of EEMS significantly inhibited tumor angiogenesis in a mouse dorsal air sac assay. In conclusion, we found that the EEMS and its resveratrol derivatives, particularly GC, suppress multiple angiogenesis-related endothelial cell functions and/or tumor angiogenesis, indicating that the melinjo seeds and the natural resveratrol derivatives may be useful for cancer prevention and treatment.

Donor age-dependent acceleration of cellular aging by repeated ultraviolet A irradiation of human dermal fibroblasts derived from a single donor.

The relationship between cellular aging and aging of entire organisms has been studied extensively.The findings are confusing, however, and no clear relationships have been demonstrated.The conflicting data may be due to individual differences among the donors of the studied cells.It is crucial to identify the changes in cellular properties that are the result of the aging process.Here, we used human dermal fibroblast cell lines established from a single donor at different ages to assess the influence of ultraviolet A (UVA) on cellular aging. These cell lines have the same genetic background and were obtained from a restricted body region. The results indicated that cellular aging was accelerated by UVA irradiation in a donor age-dependent manner. The ratio of lifespan shortening increased with donor age. Increased donor age not only decreased cell division, but also increased the growth arrest response to UVA irradiation. The characteristics of the cultured cells reflected the age-related changes in dermal fibroblasts.