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Biomedical subjects

Michael Detmar

Publications and source records attributed to Michael Detmar.

At least 55 records · Page 3Linked to original sources
PubMed2003

Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.

In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with important implications for future studies of vascular development and function.

Biomarkers↗
PubMed2003

Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.

Adult↗
PubMed2003

Thrombospondin-1 plays a critical role in the induction of hair follicle involution and vascular regression during the catagen phase.

Hair growth is associated with pronounced vascular-endothelial-growth-factor-induced perifollicular angiogenesis, whereas the catagen regression phase is characterized by apoptosis-driven blood vessel regression. The biologic relevance of endogenous inhibitors of angiogenesis in the control of hair cycling, however, has remained unknown. We studied the expression and biologic role of the angiogenesis inhibitor thrombospondin-1 (TSP-1) during the induced adult hair follicle cycle in wild-type, TSP-1 deficient, and TSP-1 overexpressing transgenic mice. TSP-1 expression was absent from hair bulb and dermal papilla cells during early to mid-anagen but was highly upregulated throughout the catagen involution phase. In TSP-1 deficient mice, the follicle growth phase was significantly prolonged, associated with increased perifollicular vascularization and vascular proliferation. Conversely, hair follicle growth was delayed in K14/TSP-1 transgenic mice that expressed high levels of TSP-1 in outer root sheath keratinocytes, associated with reduced perifollicular vascularization. These effects were most probably mediated via its antiangiogenic effects because TSP-1 did not affect the growth of cultured murine vibrissae in the absence of a functional vascular system. These results identify a critical role of TSP-1 in the induction of anagen follicle involution, with potential implications for the therapeutic modulation of hair follicle growth.

Animals↗
PubMed2003

Stroma formation and angiogenesis by overexpression of growth factors, cytokines, and proteolytic enzymes in human skin grafted to SCID mice.

Reorganization of skin during wound healing, inflammatory disorders, or cancer growth is the result of expression changes of multiple genes associated with tissue morphogenesis. We wanted to identify proteins involved in skin remodeling and select those that may be targeted for agonistic or antagonist therapeutic approaches in various disease processes. Full-thickness human skin was grafted to severe combined immunodeficient mice and injected intradermally with 38 different adenoviral vectors inserted with 37 different genes coding for growth factors, cytokines, proteolytic enzymes and their inhibitors, adhesion receptors, oncogenes, and tumor suppressor genes. Responses were characterized for infiltration of inflammatory cells, vascular density, matrix formation, fibroblast-like cell proliferation, and epidermal hyperplasia. Of the 17 growth factor vectors, 16 induced histological changes in human skin. Members of the VEGF and angiopoietin families induced neovascularization. PDGFs and TGF-betas stimulated connective tissue formation, and the chemokines IL-8 and MCP-1 attracted inflammatory neutrophils and monocytes, respectively. The serine protease uPA induced a vascular response similar to that of VEGF. Vectors with adhesion receptors, oncogenes and tumor suppressor genes had, with few exceptions, little effects on skin architecture. The overall results suggest that adenoviral vectors can effectively remodel the architecture of human skin for studies in morphogenesis, inflammatory skin disorders, wound healing, and cancer development.

Adenoviridae↗
PubMed2003

An N-terminal 80 kDa recombinant fragment of human thrombospondin-2 inhibits vascular endothelial growth factor induced endothelial cell migration in vitro and tumor growth and angiogenesis in vivo.

We have previously shown that stable overexpression of the thrombospondin-2 (TSP-2) gene inhibited the tumor growth and angiogenesis of human squamous cell carcinoma xenotransplants. To investigate the potential antitumoral efficacy of systemic TSP-2 therapy, we expressed a recombinant 80 kDa fragment of human TSP-2 (TSP-2/NTF), encompassing the N-terminal globular region through the three type 1 repeats, in human kidney 293 EBNA cells, using a modified pCEP4 expression vector. Daily intraperitoneal injections of TSP-2/NTF resulted in a significant inhibition of the growth of human A431 squamous cell carcinomas in vivo and in reduced tumor vascularization. To further investigate possible mechanisms of the antiangiogenic activity of TSP-2/NTF, several in vitro angiogenesis assays were performed in human dermal microvascular endothelial cells. TSP-2/NTF inhibited vascular endothelial growth factor induced migration of human dermal microvascular endothelial cells and inhibited tube formation on Matrigel in vitro. TSP-2/NTF also inhibited vascular endothelial growth factor induced angiogenesis in an in vivo Matrigel assay. Moreover, TSP-2/NTF potently induced human dermal microvascular endothelial cell apoptosis in vitro but did not affect A431 tumor cell proliferation or apoptosis. These findings identify TSP-2/NTF as a potent systemic inhibitor of tumor growth and angiogenesis, acting by direct inhibition of several endothelial cell functions involved in neovascularization.

Animals↗
PubMed2002

Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice.

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.

Animals↗
PubMed2002

A critical role of placental growth factor in the induction of inflammation and edema formation.

Angiogenesis is a prominent feature of a number of inflammatory human diseases, including rheumatoid arthritis, psoriasis, and cutaneous delayed-type hypersensitivity (DTH) reactions. Up-regulation of placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has been found in several conditions associated with pathologic angiogenesis; however, its distinct role in the control of angiogenesis has remained unclear. To directly investigate the biologic function of PlGF in cutaneous inflammation and angiogenesis, DTH reactions were investigated in the ear skin of wild-type mice, of PlGF-deficient mice, and of transgenic mice with targeted overexpression of human PlGF-2 in epidermal keratinocytes, driven by a keratin 14 promoter expression construct. Chronic transgenic delivery of PlGF-2 to murine epidermis resulted in a significantly increased inflammatory response, associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration than seen in wild-type mice. Conversely, PlGF deficiency resulted in a diminished and abbreviated inflammatory response, together with a reduction of inflammatory angiogenesis and edema formation. VEGF expression was up-regulated at a comparable level in the inflamed skin of all genotypes. These findings reveal that placental growth factor plays a critical role in the control of cutaneous inflammation, and they suggest inhibition of PlGF bioactivity as a potential new approach for anti-inflammatory therapy.

Animals↗
PubMed2002

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.

Animals↗
PubMed2002

Systemic inhibition of tumor growth and angiogenesis by thrombospondin-2 using cell-based antiangiogenic gene therapy.

Recent studies indicate that continuous administration improves the antitumoral efficacy of angiogenesis inhibitors, as compared with intermittent dosing, suggesting a potential role of gene therapy in antiangiogenic tumor therapy. We established a tissue-engineered implant system for the continuous in vivo production of thrombospondin-2 (TSP-2), a potent endogenous inhibitor of tumor growth and angiogenesis. Fibroblasts were retrovirally transduced to overexpress TSP-2 and were seeded onto biodegradable polymer scaffolds. After transplantation into the peritoneal cavity of nude mice, bioimplants maintained high levels of TSP-2 secretion over extended time periods, resulting in increased levels of circulating TSP-2. Bioimplant-generated TSP-2 potently inhibited tumor growth and angiogenesis of human squamous cell carcinomas, malignant melanomas, and Lewis lung carcinomas that were implanted at a distant site. These results provide the first proof-of-principle for the feasibility and therapeutic efficiency of systemic, cell-based antiangiogenic gene therapy using biodegradable polymer grafts for the treatment of cancer.

3T3 Cells↗
PubMed2002

Increased and prolonged inflammation and angiogenesis in delayed-type hypersensitivity reactions elicited in the skin of thrombospondin-2--deficient mice.

Angiogenesis and enhanced microvascular permeability are hallmarks of a large number of inflammatory diseases. Although up-regulation of proangiogenic factors such as vascular endothelial growth factor and interleukin-8 have been previously reported in inflamed tissue, the biologic role of endogenous inhibitors of angiogenesis in inflammation has remained unclear. To investigate the biologic role of the potent angiogenesis inhibitor thrombospondin-2 (TSP-2) in the control of cutaneous inflammation, delayed-type hypersensitivity reactions were elicited in the ear skin of wild-type and TSP-2-deficient mice by topical sensitization and challenge with oxazolone. Cutaneous TSP-2 expression was up-regulated in the inflamed skin of wild-type mice, predominantly in dermal fibroblasts and microvessels. Lack of TSP-2 resulted in a significantly enhanced inflammatory response with increased angiogenesis, edema formation, and inflammatory infiltration. Ear swelling and inflammation persisted for more than 2 weeks in TSP-2-deficient mice, as compared with 1 week in wild-type mice. Although baseline vascular permeability was unchanged, significantly enhanced microvascular leakage was found in the inflamed skin of TSP-2-deficient mice. Moreover, the fraction of rolling leukocytes was significantly increased in the untreated skin of TSP-2-deficient mice. These results reveal an important role of TSP-2 in limiting the extent and the duration of edema formation, angiogenesis, and inflammatory cell infiltration during acute and chronic inflammation.

Animals↗
PubMed2002

Differential effects of photoaging vs intrinsic aging on the vascularization of human skin.

OBJECTIVE: To quantify the distinct effects of photoaging vs intrinsic aging of human skin on cutaneous vascularization in the Korean population. DESIGN: Case series. SETTING: University hospital. PARTICIPANTS: A total of 21 healthy Korean volunteers from the third to the ninth decades of life. INTERVENTION: Skin biopsy specimens were obtained from chronically sun-exposed and sun-protected skin of each participant. MAIN OUTCOME MEASURES: Frozen sections were stained for the platelet endothelial cell adhesion molecule CD31 (PECAM-1), and computer-assisted quantitative image analysis was performed to quantify cutaneous vascular density and vessel size. RESULTS: Intrinsically aged and photoaged skin showed an age-dependent reduction of cutaneous vessel size. However, only photoaged skin exhibited significantly reduced numbers of dermal vessels, in particular in the subepidermal areas that displayed extensive matrix damage. Linear regression analysis revealed an inverse relation of vessel numbers and age in sun-damaged, but not in sun-protected, skin. CONCLUSIONS: In Korean skin, chronic photodamage results in a gradual decrease in the number and size of dermal vessels over several decades of sun exposure, most likely due to degenerative changes of the dermal extracellular matrix. Because the present investigation was restricted to ethnic Korean volunteers, future studies are needed to evaluate whether similar changes can be observed in whites.

Adult↗
PubMed2002

Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.

Early during development, one of the first indications that lymphangiogenesis has begun is the polarized expression of the homeobox gene Prox1 in a subpopulation of venous endothelial cells. It has been shown previously that Prox1 expression in the cardinal vein promotes and maintains the budding of endothelial cells that will form the lymphatic vascular system. Prox1-deficient mice are devoid of lymphatic vasculature, and in these animals endothelial cells fail to acquire the lymphatic phenotype; instead, they remain as blood vascular endothelium. To investigate whether Prox1 is sufficient to induce a lymphatic fate in blood vascular endothelium, Prox1 cDNA was ectopically expressed by adenoviral gene transfer in primary human blood vascular endothelial cells and by transient plasmid cDNA transfection in immortalized microvascular endothelial cells. Transcriptional profiling combined with quantitative real-time reverse transcription-polymerase chain reaction and Western blotting analyses revealed that Prox1 expression up-regulated the lymphatic endothelial cell markers podoplanin and vascular endothelial growth factor receptor-3. Conversely, genes such as laminin, vascular endothelial growth factor-C, neuropilin-1, and intercellular adhesion molecule-1, whose expression has been associated with the blood vascular endothelial cell phenotype, were down-regulated. These results were confirmed by the use of specific antibodies against some of these markers in sections of embryonic and adult tissues. These findings validate our previous proposal that Prox1 is a key player in the molecular pathway leading to the formation of lymphatic vasculature and identify Prox1 as a master switch in the program specifying lymphatic endothelial cell fate. That a single gene product was sufficient to re-program the blood vascular endothelium toward a lymphatic phenotype corroborates the close relationship between these two vascular systems and also suggests that during evolution, the lymphatic vasculature originated from the blood vasculature by the additional expression of only a few gene products such as Prox1.

Cells, Cultured↗
PubMed2002

Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth.

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by alpha-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

Angiopoietin-1↗
PubMed2002

The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis.

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.

Animals↗
PubMed2002

Stable expression of angiopoietin-1 and other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo.

Pericytes have been difficult cells to study because they do not maintain their characteristic phenotype in vitro, and they begin to express fibroblast markers after only a few days in culture. We now report methods for the isolation, purification, culture, and repurification of human dermal pericytes from mixed cell populations using an immunoaffinity-magnetic bead approach coupled with the 3G5 IgM monoclonal antibody that is specific for a pericyte surface ganglioside. These purified cells could be expanded in culture, and they maintained their pericyte phenotype for up to 8 days. In addition, they strongly expressed angiopoietin-1 (Ang-1) but not angiopoietin-2, Tie-1, or Tie-2; in contrast, dermal microvascular endothelial cells exhibited a reciprocal expression pattern. These findings are important because the close proximity of endothelial cells and pericytes has often made it difficult to determine with certainty the specific cell type(s) that expressed each of these proteins in situ. Extending our in vitro findings to two models of angiogenesis in vivo, we demonstrated a subpopulation of Ang-1-expressing cells that appeared in maturing microvessels during later stages of cutaneous wound healing and vascular permeability factor/vascular endothelial growth factor-induced angiogenesis. Our results provide strong evidence that Ang-1 is expressed by pericytes in vitro and in vivo and that the role proposed for Ang-1 in vessel maturation in development can be extended to vessel maturation after angiogenesis in adult tissues.

Adenoviridae↗
PubMed2002

Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.

Chronic ultraviolet-B irradiation of the skin results in epidermal hyperplasia, degradation of extracellular matrix molecules, and formation of wrinkles. To characterize the biologic role of the vascular system in the mediation of ultraviolet-B-induced skin damage, we performed quantitative analyses of cutaneous blood vessels of mice after 10 wk of ultraviolet-B irradiation. Skin vascularization was greatly increased after chronic ultraviolet-B exposure with a significant increase of both the number and the size of dermal blood vessels, associated with upregulation of vascular endothelial growth factor expression in the hyperplastic epidermis. To directly study whether inhibition of angiogenesis may diminish ultraviolet-B-induced cutaneous damage, wild-type and transgenic mice with skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 were subjected to the same ultraviolet-B irradiation regimen. Ultraviolet-B-irradiated thrombospondin-1 transgenic mice showed a significantly reduced skin vascularization, decreased endothelial cell proliferation, and increased endothelial cell apoptosis rates, compared with wild-type mice. Moreover, dermal photo-damage and wrinkle formation were greatly reduced in thrombospondin-1 transgenic mice. These results reveal an important role of the cutaneous vascular system in mediating ultraviolet-B-induced skin damage and suggest inhibition of angiogenesis as a potential new approach for the prevention of chronic cutaneous photo-damage.

Animals↗