Systemic sclerosis stimulates angiogenesis in the chick embryo chorioallantoic membrane

Skin biopsies from patients with systemic sclerosis (SSc) were investigated for their angiogenic activity by using the chick embryo chorioallantoic membrane (CAM) assay. Ten samples of SSc and 10 of normal skin from age- and sex-matched subjects were grafted onto the CAM, and the angiogenic response in pathological and control implants was assessed on histological sections by a planimetric point-count method 4 days after grafting. The vascular counts in the area underlying the SSc were significantly higher than those of normal skin and a dense mononuclear cell infiltrate was detectable around the blood vessels in pathological specimens. These results suggest that SSc may promote angiogenesis, perhaps leading to the release of several angiogenic factors. Moreover, the role played in the angiogenic response by the inflammatory cells forming the cellular infiltrate is suggested by this study.     

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

AIMS/HYPOTHESIS: We studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates ( db+/+m). METHODS: Animals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds. RESULTS: We found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strength of the wound and enhanced the wound content of VEGF. CONCLUSION/INTERPRETATION: Our study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.     

Akt1 is necessary for the vascular maturation and angiogenesis during cutaneous wound healing

Previous in vivo and in vitro studies have shown that Akt1 serves as a crucial regulator of vascular maturation, extracellular matrix composition, and angiogenesis in tumors. Hence, we hypothesized that Akt1 may be necessary for other angiogenesis-dependent processes, including wound healing. Using Akt1 (-/-) and Akt2 (-/-) mice, we demonstrate that deficiency of Akt1, but not Akt2, results in impaired assembly of collagen in skin wounds and around the blood vessels. Wounds in Akt1 (-/-) mice, but not in Akt2 (-/-) mice, were characterized by reduced vascular area as well as impaired vascular maturation as evidenced by reduced smooth muscle cell recruitment. Expression level of a major angiogenic growth factor, VEGF, was significantly lower in wound tissues of Akt1 (-/-) mice as compared to WT. However, despite the impaired collagen assembly and reduced angiogenesis in Akt1 (-/-) wounds, no significant difference in migration of fibroblasts into the wound area was observed between WT and Akt1 (-/-) mice. Importantly, the dynamics of wound closure were similar between WT, Akt1 (-/-), and Akt2 (-/-) mice. Thus, it appears that although the lack of Akt1 impairs VEGF expression, wound angiogenesis, and subsequent maturation of vasculature, it has no effect on the wound closure. These findings may have clinical applications for the improvement of treatment procedures with reported history of wound healing complications.     

Endogenous and exogenous fibroblast growth factor-2 modulate wound healing in the chick embryo chorioallantoic membrane

Re-epithelization and the formation of a granulation tissue consisting of inflammatory cells, newly formed blood vessels, and fibroblasts embedded in a loose collagenous extracellular matrix, are critical events occurring during wound healing. In this study, utilizing the chick embryo chorioallantoic membrane (CAM) as an in vivo model of wound healing, we investigated the role of endogenous and exogenous fibroblast growth factor-2 (FGF-2) in the wound healing reparative processes. The results showed that: (1) neutralizing anti-FGF-2 antibodies (400 ng/embryo) decreased significantly the rate of wound healing (occurring only in 25% of specimens) when applied close to the edge of the wound, causing a significant decrease of microvessel and fibroblast density, and of an inflammatory macrophage infiltrate in the wounded area; (2) conversely, the application of exogenous recombinant FGF-2 (1.0 μg/embryo) greatly accelerated the wound repair occurring approximately 24h earlier than in untreated CAMs, stimulating angiogenesis, fibroblast proliferation, and macrophage infiltration. These findings demonstrate the role of FGF-2 in wound healing of the CAM and suggest that CAM, usually employed as an in vivo assay to study angiogenesis, can also be utilized as an in vivo model for the easy, rapid, and economic screening of molecules potentially able to affect the wound healing process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Localization and regulation of pregnancy-associated plasma protein a expression in healing human skin

Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with alpha-smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.     

Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 micrograms of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cell-mediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.     

Growth regulation of skin cells by epidermal cell-derived factors: implications for wound healing.

Epidermal cell-derived factors (EDF), present in extracts and supernatant fluids of cultured epidermal cells, were found to stimulate the proliferation of keratinocytes but to inhibit fibroblasts. In vitro, the effect of EDF on epidermal cells resulted in an increased number of rapidly proliferating colonies composed mainly of basal keratinocytes. Control cultures grown in the absence of EDF had a high proportion of terminally differentiated cells. In fibroblast cultures EDF inhibited the ability of fibroblasts to cause contraction of collagen sponges by 90%. Epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, transforming growth factor beta, nerve growth factor, and extracts of WI-38 cells (human embryonic lung fibroblasts) did not have this inhibitory activity. Application of EDF to surgical wounds stimulated extensive migration and proliferation of keratinocytes from remnants of glands, hair follicles, and wound edges. The restoration of complete epidermal coverage of wounds treated with EDF occurred twice as rapidly as that of control wounds. In addition, regenerating dermis in the EDF-treated wounds contained 1/5th to 1/15th as many cells as wounds treated with epidermal growth factor, urogastrone, transforming growth factor, or phosphate-buffered saline. The use of EDF to enhance re-epithelization and to prevent scar formation is proposed.     

Increased expression of angiogenic factors in human colonic angiodysplasia

OBJECTIVE: Angiodysplasia of the colon is a distinct vascular abnormality characterized by focal accumulation of ectatic vessels in the mucosa and submucosa. To investigate whether angiogenesis contributes to the pathogenesis of human colonic angiodysplasia, we examined the expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and its endothelial cell receptors flt-1 and KDR. METHODS: Immunohistochemistry was performed in sections of specimens obtained from 18 patients with colonic angiodysplasia and from eight patients with colon cancer and its adjacent, histologically normal margins of resection. We used affinity-purified rabbit polyclonal antibodies and a streptoavidin-biotin peroxidase method. RESULTS: We detected strong immunoreactivity for vascular endothelial growth factor, homogeneously distributed in the endothelial lining of blood vessels of all sizes in 16 (89%) specimens of colonic angiodysplasia and in seven (88%) patients with colon cancer. In contrast, very limited immunoreactivity was found in normal colon. Vascular staining for flt-1 was observed in eight (44%) and one (12.5%) of the colonic angiodysplasia or colon cancer specimens, respectively, but not in normal colon. Vascular immunoreactivity for basic fibroblast growth factor was observed in seven (39%) specimens from patients with colonic angiodysplasia, whereas either very limited or no immunostaining was found in sections from specimens of patients with colon cancer and its normal margins. CONCLUSIONS: In human colonic angiodysplasia, increased expression of angiogenic factors is likely to play a pathogenic role.     

AMP-activated protein kinase signaling stimulates VEGF expression and angiogenesis in skeletal muscle

AMP-activated protein kinase (AMPK) is regulated by various cellular stresses. Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is also upregulated by several stress-inducible factors such as hypoxia and stimulation by cytokines and growth factors. Here, we investigated whether AMPK signaling in muscle has a role in regulating VEGF-mediated angiogenic processes. AICAR stimulated VEGF mRNA and protein levels in C2C12 myotube cultures. Transduction with dominant-negative AMPK abolished AICAR-induced VEGF expression at both steady state mRNA and protein levels. AICAR increased VEGF mRNA stability without affecting VEGF promoter activity. AICAR also stimulated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Activation of p38 MAPK was suppressed by transduction with dominant-negative AMPK, suggesting that AMPK is upstream of p38 MAPK. The p38 MAPK inhibitor SB203580 blocked AICAR-induced increase in VEGF mRNA and protein levels, indicating that AICAR-mediated VEGF induction is dependent on p38 MAPK signaling. AICAR treatment increased VEGF expression and accelerated angiogenic repair of ischemic hindlimbs in mice in an AMPK-dependent manner. These data indicate that AMPK-p38 MAPK signaling cascade can increase VEGF production in muscle and promote angiogenesis in response to ischemic injury.     

Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells.

The identity of the paracrine mediator(s) of the antiresorptive action of estrogen on bone cells is controversial. Osteoprotegerin (OPG) was recently identified as a soluble member of the tumor necrosis factor (TNF) receptor (TNF-R) superfamily that is secreted by osteoblast lineage cells and acts by binding to and neutralizing its cognate ligand, OPG-L, a required factor for osteoclastogenesis. OPG prevents bone loss when administered to ovariectomized rats, induces osteoporosis when ablated in knock-out mice, and induces osteopetrosis when overexpressed in transgenic mice. In conditionally immortalized, human osteoblastic hFOB/ER-3 and hFOB/ER-9 cell lines containing physiological concentrations of approximately 800 and approximately 8,000 functional estrogen receptors (ER)/nucleus, respectively, we found that 17beta-estradiol dose- and time-dependently increased OPG mRNA and protein levels to maximal levels of 370% and 320%, respectively (P < 0.001); co-treatment with the "pure" antiestrogen ICI 182,780 abrogated these effects completely. 17beta-Estradiol also dose-dependently increased OPG mRNA and protein levels in normal human osteoblasts with approximately 400 ER/nucleus by 60% and 73%, respectively. Thus, estrogen enhancement of OPG secretion by osteoblastic cells may play a major role in the antiresorptive action of estrogen on bone.     

The periphery of the human fetal adrenal gland is a site of angiogenesis: zonal differential expression and regulation of angiogenic factors

CONTEXT: Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. OBJECTIVE: Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. DESIGN: Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. MAIN OUTCOME MEASURES: Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. RESULTS: Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. CONCLUSION: Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.     

Lactate stimulates angiogenesis and accelerates the healing of superficial and ischemic wounds in mice

Wounds notoriously accumulate lactate as a consequence of both anaerobic and aerobic glycolysis following microcirculation disruption, immune activation, and increased cell proliferation. Several pieces of evidence suggest that lactate actively participates in the healing process through the activation of several molecular pathways that collectively promote angiogenesis. Lactate indeed stimulates endothelial cell migration and tube formation in vitro, as well as the recruitment of circulating vascular progenitor cells and vascular morphogenesis in vivo. In this study, we examined whether the pro-angiogenic potential of lactate may be exploited therapeutically to accelerate wound healing. We show that lactate delivered from a Matrigel matrix improves reperfusion and opposes muscular atrophy in ischemic hindlimb wounds in mice. Both responses involve lactate-induced reparative angiogenesis. Using microdialysis and enzymatic measurements, we found that, contrary to poly-L-lactide (PLA), a subcutaneous implant of poly-D,L-lactide-co-glycolide (PLGA) allows sustained local and systemic lactate release. PLGA promoted angiogenesis and accelerated the closure of excisional skin wounds in different mouse strains. This polymer is FDA-approved for other applications, emphasizing the possibility of exploiting PLGA therapeutically to improve wound healing.     

Penicillamine in rheumatoid arthritis: wound healing, skin thickness and osteoporosis

D-Penicillamine alters the normal metabolism of collagen by inhibiting cross-linking and protein synthesis. This could affect wound healing, accelerate skin thinning and possibly exaggerate the osteoporosis of rheumatoid disease. The mean time to wound healing after 42 orthopaedic surgical operations in 21 patients treated with penicillamine was 19.8 (+/- 13.1) days. Compared with an earlier study, these results suggest that the drug has a comparable effect on would healing to corticosteroids given for three years. Skinfold thickness over the fourth metacarpal of the dominant hand was measured in 28 cases before and during penicillamine treatment. There was a significant decrease both in the first and second four-month periods of treatment (P less than 0.005 and P less than 0.01). Corticosteroids in constant dose did not have an additive effect. In view of the wound healing findings the significance of these results must await further sequential measurements. The normal progression of osteoporosis over three years was documented in 70 patients who had not received penicillamine. Penicillamine reversed this trend in 35 patients after one year of treatment (P less than 0.005). The results confirm that the osteoporosis is related to disease severity rather than drug therapy.     

Different susceptibilities of chick embryo liver cells in vitro to aflatoxin,actinomycin D,and mitomycin C

Summary In the presence of aflatoxin, parenchymal cells of chick embryo liver were found to be more susceptible than mesenchymal cells, the toxic effect being an inhibition of RNA synthesis followed by protein and DNA synthesis studied by autoradiography. Actinomycin D, contrary to aflatoxin, acted on the mesenchymal cells more strongly than on the parenchymal cells. RNA synthesis of both cell types was not disturbed initially by mitomycin C, though later there was a moderate action on RNA and protein synthesis of parenchymal cells and subsequently also on those mesenchymal cells.

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Die unterschiedliche Empfindlichkeit von Zellen der embryonalen Hühnerleber in vitro gegenüber Aflatoxin, Actinomycin D und Mitomycin C

Zusammenfassung Bei Anwesenheit von Aflatoxin erwiesen sich die Parenchymzellen der embryonalen Hühnerleber empfindlicher als die mesenchymalen Zellen; die toxische Wirkung der Substanz drückte sich in einer mittels Autoradiographie festgestellten Behinderung der RNS-Synthese aus, die von Protein- und DNS-Synthese gefolgt war. Im Gegensatz zuAflatoxin wirkte Actinomycin D auf die Mesenchymzellen stärker als auf die Parenchymzellen. Durch Mitomycin C wurde die RNS-Synthese beider Zelltypen anfänglich nicht gestört, doch war später eine mäßige Wirkung auf die RNS- und Protein-Synthese der Parenchymzellen und weiterhin auch der Mesenchymzellen nachweisbar.

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The authors wish to thank Prof. T. Arai of the Institute of Food Microbiology for providing the aflatoxin and actinomycin D used in these studies.

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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Welfare.     

分泌表达PR39重组腺相关病毒对缺氧鸡胚促血管生成的影响

目的 探讨腺相关病毒(AAV)介导的融合基因NT4-TAT-His-PR39对缺氧人脐静脉内皮细胞株ECV304内缺氧诱导因子1α(HIF-1α)表达的影响及其对缺氧环境鸡胚血管生成的影响.方法 PCR技术和T载体克降法克隆PR39基因,将编码PR39、穿膜肽TAT、6×His及NT4信号肽四个基因片段相连.磷酸钙沉淀法获得重组携带NT4-TAT-His-PR39的从V载体,然后AAV-PR39感染ECV304,免疫细胞化学检测ECV304胞内6×His表达情况.在1%O2条件培养ECV304,免疫细胞化学测定AAV-PR39组和PBS组胞内HIF-1α蛋白表达.30只鸡胚随机分为从V-PR39组、EV组和PBS组(各组n=10),分别在鸡胚尿膜囊(CAM)上加AAV-PR39、EV、PBS,然后每组各取5只鸡胚分别在低氧(5%O2)和常氧(21%O2)条件培养.图像软件Image Pro Plus(IPP)分析CAM血管密度.结果 AAV-PR39组ECV304中检测到6×His表达.低氧环境下,AAV-PR39组ECV304中HIF-1α蛋白表达明显高于PBS组,AAV-PR39组CAM血管密度明显大于其他两组.结论重组携带NT4-TAT-His-PR39的AAV载体能在ECV304中分泌表达,并提高缺氧细胞HIF-1α蛋白表达水平.重组AAV载体显著促进缺氧CAM血管生成,而对常氧CAM无此作用.     

In vitro angiogenic potency in human microvascular endothelial cells derived from myocardium, lung and skin

Human microvascular endothelial cells derived from myocardium (HCMEC), lung (HPMEC) and foreskin (HDMEC) showed different angiogenic potency when cultivated in their original growth media provided by the distributors. In order to standardize microenvironmental conditions in an all-in-one assay of angiogenesis the aim of this study was to find one optimal growth medium for the endothelial cells derived from the different organs. Therefore each endothelial cell type was cultivated under identical conditions in the different original growth media as well as in several media formulations of the original growth media. Results reveal that even if cultivated in the same growth medium under exactly the same cultivation conditions--over a prolonged time period of 60 days--the endothelial cells still showed different angiogenic potency. This is due to a combination of extrinsic factors, i.e. the isolation procedure and in particular the growth medium, as well as to intrinsic differences between cells of diverse origin.     

Impaired tumor growth,metastasis, angiogenesis and wound healing in annexin A1-null mice

Despite 2 decades of research, no clear function for annexin A1 (AnxA1) has been established. Using AnxA1-KO mice, we show that tumor growth and metastasis are significantly decreased, whereas rodent survival and tumor necrosis are greatly increased when tumors grow in AnxA1-KO mice. Systems analysis of gene expression in these tumors specifically implicates 2 related vascular functions, angiogenesis and wound healing, in this impairment. Both tumor vascular development and wound healing are greatly retarded in KO tissues. Aortic ring assays reveal induced AnxA1 expression on sprouting endothelial cells of normal mice whereas KO aortas exhibit impaired endothelial cell sprouting that is rescued by adenoviral expression of AnxA1. Key differences in specific gene regulation may define new molecular pathways mediating angiogenesis, including a reset profile of pro- versus anti-angiogenic factors, apparently distinct for physiological versus pathological angiogenesis. These studies establish novel pro-angiogenic functions for AnxA1 in vascular endothelial cell sprouting, wound healing, and tumor growth and metastasis, thereby uncovering a new functional target for repairing damaged tissue and treating diseases such as cancer. They also provide critical new evidence that the tumor stroma and its microenvironment can greatly affect tumor progression and metastasis.