HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/12/1689 04-3647fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vihanto, M. M. Articles by Huynh-Do, U. Search for Related Content PubMed PubMed Citation Articles by Vihanto, M. M. Articles by Huynh-Do, U.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin

Meri M. Vihanto^*, Jan Plock, Dominique Erni, Brigitte M. Frey^*, Felix J. Frey^* and Uyen Huynh-Do^*,1

^* Department of Nephrology and Hypertension, Department of Clinical Research, and
Department of Plastic Surgery, Inselspital, University of Bern, Bern, Switzerland

¹ Correspondence: Department of Nephrology and Hypertension, University of Bern, CH-3010 Bern, Switzerland. E-mail: uyen.huynh-do{at}insel.ch

SPECIFIC AIMS

Eph receptors and ephrins are key players during the development of the embryonic vasculature, but their role in adult angiogenesis remains to be defined. We addressed the hypothesis that hypoxia might be an important mediator linking Ephs/ephrins expression to neovascularization,

1. We developed a mouse model of segmental skin hypoxia
This model allowed us to assess histopathological changes and gene expression patterns of hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), and Ephs/ephrins under hypoxic conditions.

2. We used RNA interference against HIF-1 to confirm the key role of HIF-1 in the hypoxia-dependent up-regulation of Ephs and ephrins

PRINCIPAL FINDINGS

1. Validation of the mouse dorsal skin flap model of segmental hypoxia
The dorsal skin flap pedicled on a branch of the deep circumflex iliac vessels on each side was designed so that hypoxia occurred in the distant, cranially located areas without leading to subsequent necrosis. The partial oxygen tension and microdialysis probes were inserted into the healthy neck skin (control), the well-vascularized caudal intermediate part of the flap, and the hypoxic cranial part of the flap. The hypoxic part of the flap showed significantly lower mean levels (±SEM) of partial cutaneous oxygen tension (3.44±0.12 mmHg) compared with its intermediate part (17.3±0.31 mmHg) or normal skin (27.3±0.22 mmHg) (n=8, P<0.05, Fig. 1 A, upper panel), and the mean lactate/pyruvate ratio was significantly increased in the hypoxic part of the flap (80.4±2.14) compared with the intermediate part (26.4±0.84) or normal skin (18.6±0.68) (n=8, P<0.05, Fig. 1A , top) as determined by microdialysis every 30 min for a period of 8 h.

View larger version (41K): [in this window] [in a new window]   Figure 1. A) Partial cutaneous oxygen tension measurements and microdialysis analysis. Upper panel: partial cutaneous oxygen tension is monitored continuously in the hypoxic and the intermediate parts of the flap, as well as in normal skin in 8 animals. Lower panel: microdialysis samples are taken hourly from all three parts of the skin. B) mRNA and protein expression of HIF-1 in dorsal skin after exposure to hypoxia. Basal reference levels in control skin are defined as 1 (mean±SD, n=4). C) Immunofluorescence staining for HIF1 (green signal) in hypoxic (1) and normal (2) skin (600x).

2. HIF-1 accumulates after hypoxia induction in the skin
The oxygen-dependent -subunit of HIF-1 showed maximal mRNA levels after 6 h hypoxia (6.4-fold compared with normal skin, n=4, Fig. 1B , top), as detected by real-time PCR. The protein expression increased steadily up to 24 h (6.9-fold, n=4, Fig. 1B , bottom) after hypoxia and decreased thereafter, as analyzed by Western blot. These findings were strongly corroborated by immunofluorescence studies 24 h after exposure to hypoxia: in the hypoxic skin we demonstrated a strong accumulation of HIF-1 in endothelial and adjacent cells (Fig. 1C1 ); no HIF-1 staining was observed in normal skin (Fig. 1C2 ).

3. Target gene VEGF is up-regulated in dermal endothelial cells by HIF-1
Vascular endothelial growth factor, a strong stimulator of angiogenesis and well-established target gene for HIF-1, was up-regulated in a similar fashion. The highest VEGF mRNA levels were observed after 6 h of hypoxia exposure (8.5-fold compared with normal skin). Up-regulation of VEGF protein reached its peak after 24 h of hypoxia (4.3-fold); levels were still high after 48 h (3.7-fold). These results were confirmed by immunohistochemistry analysis, where VEGF expression was strongly up-regulated in dermal endothelial cells 24 to 48 h after hypoxia.

4. Histological analysis of the hypoxic skin flap shows inflammation and endothelial cell proliferation
Inflammatory response due to hypoxia was indicated by the presence of fibrinogen in the epidermis as well as by lymphomonocytic infiltration of the subcutis after 48 h, and by the disruption of the affected vessels in the hypoxic skin. The number of proliferating endothelial cells in dermal vessels increased significantly 24 h after hypoxia (4.4-fold) and was even higher after 48 h (18-fold) (P<0.05) compared with normal skin, using K_i-67 as a cell proliferation marker.

5. EphB4, ephrinB2, EphA2, and ephrinA1 are up-regulated in the hypoxic skin
Real-time PCR and immunoblotting analyses demonstrated up-regulation of mRNA and protein expression of Ephs and ephrins of both A and B subclasses in the hypoxic mouse skin. The most prominent changes were seen for EphB4 receptor and ephrinA1 ligand. EphB4 mRNA expression was up-regulated after 12 h of hypoxia (5.8-fold compared with normal skin), on the protein level, up-regulation reached its maximum (10-fold) after 24 h. EphrinA1 mRNA expression peaked after 24 h of hypoxia (5-fold compared with normal skin) and the protein was strongly up-regulated in a similar fashion after 24 h of hypoxia (19.8-fold). In agreement with the gene expression data, immunohistochemical analysis confirmed the up-regulation of EphB4, ephrinB2, EphA2, and ephrinA1 24 h after exposure to hypoxia (Fig. 2 ). After 48 h, expressions were lower than at 24 h but still higher than in normal skin.

View larger version (111K): [in this window] [in a new window]   Figure 2. A) EphB4 (a), ephrinB2 (b), EphA2 (c), ephrinA1 (d), and control with nonimmune rabbit serum (e) expression (brown signal) shown by immunohistochemistry analysis in normal skin (1), 24 h (2) and 48 h after (3) exposure to hypoxia (400x), illustrating the localization of the proteins of interest Counterstaining with hematoxyline and development with DAB. Vessels are indicated by arrowheads. B) Semiquantitative scoring analysis of dorsal skin (values are means of particle analysis counts calculated from 100x magnification pictures in supplementary Fig. 1 by Image J software, mean±SD, *P<0.05 in all columns).

6. RNA interference for HIF-1 inhibits the hypoxia-dependent up-regulation of Eph receptors/ephrins expression
To directly demonstrate that up-regulation of Eph receptors/ephrins expression by hypoxia is indeed mediated by HIF-1, we performed chemical hypoxia experiments with cobalt chloride (CoCl₂) and knocked down HIF-1 using RNA interference. Under normoxic conditions, transfection of Hep3B and PC-3 cells with siRNA against HIF-1 had no effect on basal mRNA expression of HIF-1 or VEGF compared with nontransfected cells. The HIF-1 mRNA was up-regulated 7.6- and 6-fold in PC-3 and Hep3B cells, respectively after 8 h of CoCl₂ treatment. The VEGF mRNA was up-regulated in a similar fashion after 20 h of chemical hypoxia in both cells types: PC-3 cells showed a 5.3-fold up-regulation whereas in Hep3B cells up-regulation was 7.4-fold. CoCl₂-induced up-regulation of HIF-1 and VEGF was clearly abrogated by siRNA against HIF-1, whereas random siRNA had no effect. The effect of HIF-1 knockdown on EphA2 and ephrinA1 was assessed in PC-3 cells; for EphB4 and ephrinB2 we used Hep3B cells. The prominent reduction (60–95%) of relative mRNA levels of EphB4, ephrinB2, EphA2, and ephrinA1 after transfection with siRNA against HIF-1 was clearly confirmed at the protein level by immunofluorescence experiments.

CONCLUSIONS AND SIGNIFICANCE

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a multistep process involving a diverse array of molecular signals to stimulate endothelial cell proliferation, migration, and assembly. Endothelial cell receptor tyrosine kinases (RTKs) have been recognized as critical mediators of angiogenesis: these are the VEGF receptor, Tie, and Eph RTKs, which, together with their ligands, are key players during the development of the embryonic vasculature. The function of both VEGF/VEGF receptor and angiopoietins/Tie-2 receptor families in vascular development and angiogenesis are well studied whereas the Eph family represents a newer class of RTKs. Evidence suggests that both hypoxia and inflammatory mediators invoke an orchestration of several endothelial growth factors and receptors that play different but interacting roles to achieve an adaptation of the vasculature under conditions of impaired perfusion. Therefore, we addressed the hypothesis that the regulation of Ephs/ephrins expression could be mediated by hypoxia.

We first validated a new in vivo model allowing continuous and reliable, quantitative assessment of local segmental tissue hypoxia by measuring the partial cutaneous oxygen tension and the lactate/pyruvate ratio in the mouse skin. The considerable potential of this versatile model lies in the possibility of in situ assessment of molecular, metabolic, and histological parameters relevant for understanding local tissue hypoperfusion causing a defined degree of hypoxia. This model is highly reproducible due to the orientation of the flap design on the local anatomic features and allows us to investigate any gene of interest in transgenic mice. We proved that the model truly represents segmental skin hypoxia, by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of HIF-1 induction, and immunofluorescence localization of stabilized HIF-1 in the hypoxic flap.

Next we showed that hypoxia is a strong stimulus for Eph receptors and ephrins of both A and B subclasses. During embryonic development, ephrinB2 is an early marker of arterial endothelial cells, and EphB4 reciprocally marks venous endothelial cells. Endothelial cells in the adult maintain their asymmetric arteriovenous expression pattern, suggesting that the EphB/ephrinB system plays an important role in controlling vascular homeostasis. We demonstrated the most prominent up-regulation with EphB4 receptor and ephrinA1 ligand, an intriguing finding suggesting a complementary role of A and B subclasses of receptors and ligands in the hypoxia response. Since bi-directional signaling is a hallmark of Eph/ephrin interaction, our observation raises the possibility that this complementary pattern not only reflects a redundancy of signaling, but could also demarcate boundaries between different regions, an important issue deserving further consideration. Our findings suggest that the VEGF/VEGF receptor and ephrins/Eph receptor families act synergistically in the mouse skin to induce angiogenesis in response to local hypoxia (Fig. 3 ).

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic representation of up-regulation of Eph receptors and ephrins after hypoxia exposure. The findings presented here suggest a synergistic action of the VEGF receptor/VEGF and Eph receptors/ephrins systems in the skin in response to local tissue hypoxia. They identify HIF-1 as an important regulator of Ephs and ephrins expression.

Finally, our RNA interference studies demonstrate that HIF-1 siRNA treatment abrogates hypoxia-induced up-regulation of Eph receptors and ephrins of both subclasses. In aggregate, our findings establish HIF-1 as an important regulatory factor for Ephs/ephrins expression and present evidence for a role of Ephs/ephrins in tissue repair and wound healing, extending their function in adult angiogenesis beyond tumor neovascularization. The model presented here offers considerable potential for analyzing the mechanisms of neovascularization in the skin, an important issue in physiological and pathological angiogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3647fje; doi: 10.1096/fj.04-3647fje

This article has been cited by other articles:

				M. Lackmann and A. W. Boyd Eph, a Protein Family Coming of Age: More Confusion, Insight, or Complexity? Sci. Signal., April 15, 2008; 1(15): re2 - re2. [Abstract] [Full Text] [PDF]

				J. D. Blais, C. L. Addison, R. Edge, T. Falls, H. Zhao, K. Wary, C. Koumenis, H. P. Harding, D. Ron, M. Holcik, et al. Perk-Dependent Translational Regulation Promotes Tumor Cell Adaptation and Angiogenesis in Response to Hypoxic Stress Mol. Cell. Biol., December 15, 2006; 26(24): 9517 - 9532. [Abstract] [Full Text] [PDF]

				D. M. Brantley-Sieders, W. B. Fang, Y. Hwang, D. Hicks, and J. Chen Ephrin-A1 Facilitates Mammary Tumor Metastasis through an Angiogenesis-Dependent Mechanism Mediated by EphA Receptor and Vascular Endothelial Growth Factor in Mice Cancer Res., November 1, 2006; 66(21): 10315 - 10324. [Abstract] [Full Text] [PDF]

				M. M. Vihanto, C. Vindis, V. Djonov, D. P. Cerretti, and U. Huynh-Do Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase J. Cell Sci., June 1, 2006; 119(11): 2299 - 2309. [Abstract] [Full Text] [PDF]

				T. Korff, G. Dandekar, D. Pfaff, T. Fuller, W. Goettsch, H. Morawietz, F. Schaffner, and H. G. Augustin Endothelial EphrinB2 Is Controlled by Microenvironmental Determinants and Associates Context-Dependently With CD31 Arterioscler. Thromb. Vasc. Biol., March 1, 2006; 26(3): 468 - 474. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/12/1689 04-3647fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vihanto, M. M. Articles by Huynh-Do, U. Search for Related Content PubMed PubMed Citation Articles by Vihanto, M. M. Articles by Huynh-Do, U.