VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting
TetON-HIF-1:VEGF Mice Lack Angiogenesis and Vessel Remodeling.
To test the role of VEGF during epithelial HIF-1–induced neovascularization, we combined TetON-HIF-1 mice (11) with K14-Cre transgenic and VEGF floxed (VEGF) knock-in mice (12–15). The final genotype of these composite mice was K14-rtTA:TRE-HIF-1α:K14-Cre:VEGF. This genotype, designated as TetON-HIF-1:VEGF, was n = 12 in the FVB/n strain. Doxycycline (DOX) treatment of 8- to 12-wk-old TetON-HIF-1:VEGF mice induced a robust neovascularization that peaked and plateaued 14 d after initiation (Fig. 1 A and B). In contrast, neovascularization failed to occur in TetON-HIF-1:VEGF composite mice (Fig. 1 A and B). Moreover, contrary to prior reports (16), there was no reduction of microvascular density in TetON-HIF-1:VEGF composite mice, at baseline before DOX induction compared with nontransgenic controls, likely due to differences in tissue fixation/epitope preservation techniques and endothelial marker antibody detection reagents. Induction of HIF-1α protein expression was detectable in both interfollicular epidermal and hair follicle outer root sheath basal keratinocytes in both TetON-HIF-1:VEGF and TetON-HIF-1:VEGF mice after 14 days of DOX induction (Fig. 1C).
Temporal control of HIF-1 allowed us to deploy an emerging technology for noninvasive and label-free functional determination of microvascular dynamics, optical-resolution photoacoustic microscopy (OR-PAM) (17, 18). The resolving power and image segmentation capabilities of OR-PAM enabled multiparameter determination of vascular morphology including capillary volume as an indicator of angiogenesis, and arteriovenous (arteriolar/venular) volume, vessel length, diameter, and tortuosity as measures of vascular remodeling. Because OR-PAM uses hemoglobin for contrast (17), both the imaging and quantitative segmentation analysis were based on perfused vessels, which, similar to ante mortem lectin perfusion (10), is distinct from data derived from endothelial marker analysis of postmortem tissue sections. Serial OR-PAM monitoring of individual mice from days 0 to 60 of continuous DOX provision revealed that HIF-1 activation in TetON-HIF-1:VEGF mice produced an eightfold increase in capillary volume and a 3.5-fold elevation of arteriovenous and overall vascular volume compared with DOX-induced TetON-HIF-1:VEGF or day 0 TetON-HIF-1:VEGF controls (Fig. 2A and Fig. S1B). OR-PAM segmentation analysis suggested that capillary volume fell between days 14 and 30 in the TetON-HIF-1:VEGF mice (Fig. S1). The difference between these data and the microvessel density analysis based on CD31 decoration of all vessels could be due to the lack of perfusion of CD31-positive vessels or that OR-PAM acquires wide field volumetric data and immunofluorescence analyzes microvascular area in 2D tissue sections. OR-PAM also demonstrated abrogation of HIF-1–mediated vasodilatation and tortuosity induction in the absence of epithelial VEGF (Fig. S1B).
Loss of HIF-1–Induced VEGF Produces Sprouting Without Endothelial Proliferation.
An outstanding feature of the TetON-HIF-1 model is the ability to dissect discrete stages of neovascular network development and its maintenance due to temporal control of HIF-1 gain of function (11). Here, we tested the necessity of VEGF for HIF-1 induction of the earliest stages of neovascularization, endothelial sprouting, and proliferation. DOX-treated NTG and day 0 TetON-HIF-1:VEGF mice were used interchangeably in all studies, because they were phenotypically and functionally similar. Endothelial proliferation remained at barely detectable levels after HIF-1 activation in TetON-HIF-1:VEGF mice, in contrast to a robust induction in TetON-HIF-1:VEGF counterparts (Fig. S2A). Surprisingly, TetON-HIF-1:VEGF mice evidenced a prolonged interval of endothelial sprouting up to day 14 during HIF-1 induction (Fig. 3A, Left and Fig. S2B). In contrast, sprout elaboration in TetON-HIF-1:VEGF was restricted to the immediate postinduction interval, markedly evident at DOX day 3, but nearly undetectable by DOX day 14 (Fig. 3A, Left and Fig. S2B). Sprouts were not detectable in NTG mice at any interval during DOX treatment (Fig. 3A, Left and Fig. S2B). Because Dll4-Notch1 activation regulates vascular sprouting, and is downstream of VEGF signaling via VEGFR2 and VEGFR3 (19–21), we interrogated mouse ear cross-sections for dual Dll4/CD31 coimmunofluorescence (Fig. 3A, Right). Endothelial Dll4 expression levels in TetON-HIF-1:VEGF mice were low and similar to those of their NTG counterparts up to 30 d of continuous HIF-1 induction (Fig. 3A, Right). In contrast, TetON-HIF-1:VEGF mice evidenced marked endothelial Dll4 up-regulation after 14 d of HIF-1 induction (Fig. 3, Right Middle). We further explored Notch-1 signaling by determining the expression kinetics and localization of the Notch intracellular domain (NICD), the product of the gamma secretase-mediated receptor cleavage and the active transcriptional Notch component (22). The NICD was detectable in both the day 14 TetON-HIF-1:VEGF endothelium and in basal keratinocytes (Fig. 3B, arrowheads). In contrast, the NICD was detectable only in a few basal keratinocytes of TetON-HIF-1:VEGF mice. The levels of Dll4 and the downstream Notch target Hey 1 mRNA were elevated 7- to 10-fold in TetON-HIF-1:VEGF mice compared with 14-d DOX-treated NTG controls (Fig. 3C). Dll4 and Hey-1 were induced at low, but statistically significant, levels in TetON-HIF-1:VEGF mice compared with NTG controls (Fig. 3C). Collectively, these data suggest that HIF-1 and its downstream targets (see below) are able to induce endothelial sprouting, yet these sprouts are nonproductive in forming neovessels in the absence of VEGF. The paucity of epidermal NICD in the TetON-HIF-1:VEGF mice demonstrates that VEGF is required for robust Notch activation in this cellular tissue compartment after HIF-1 induction.
Induction of Multiple HIF-1 Angiogenic Target Genes Is Insufficient for Angiogenesis in the Absence of VEGF.
Previous work has highlighted the angiogenic potential of several direct and indirect HIF-1 transcriptional targets (23–29). To determine whether these targets were induced by HIF-1 in the absence of VEGF, whole skin extracts were analyzed by using RT-PCR, ELISA, or antibody microarrays (Fig. 4 A and E). First, we determined the functional efficiency of keratin-14 regulated Cre-mediated VEGF deletion. Both mRNA and whole tissue ELISA revealed baseline or even lower VEGF levels, compared with DOX-treated NTG controls, despite HIF-1 induction.
Other targets exhibited four expression patterns. PIGF, angiogenin, MMP-9, PAI-1, and MMP-3 were up-regulated in TetON-HIF-1:VEGF mice at levels comparable with TetON-HIF-1:VEGF controls (Fig. 4F and Fig. S3 A and B). Adrenomedullin (ADM) and iNOS expression were lower in TetON-HIF-1:VEGF mice on DOX day 3, increased during continuous HIF-1 activation for 14 and 30 d, yet did not reach the levels evident in TetON-HIF-1:VEGF positive controls (Fig. 4 B and C). Carbonic anhydrase IX (CAIX) progressively increased in TetON-HIF-1:VEGF mice to levels comparable with TetON-HIF-1:VEGF positive controls by 30 d of continuous HIF-1 induction (Fig. 4D). The fourth expression pattern tracked with VEGF expression, lack or markedly reduced induction in TetON-HIF-1:VEGF mice compared with marked up-regulation in TetON-HIF-1:VEGF counterparts. These molecules, osteopontin (OPN), pentraxin-3 (PTX-3), cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed (CCN3/NOV), and macrophage chemoattractant protein-1 (MCP-1), are expressed in activated inflammatory and stromal fibroblasts (30–32). Their expression pattern was consistent with the marked diminution in stromal myeloid cell recruitment and retention in TetON-HIF-1:VEGF compared with TetON-HIF-1:VEGF mice (see below).
Epithelial VEGF Loss Markedly Impairs HIF-1–Mediated Myeloid Cell Recruitment.
HIF-1 is known to induce myeloid cell mobilization, recruitment, and retention via VEGF, PlGF, and SDF1 (11, 33–36). As such, we determined myeloid cell recruitment to HIF-1 stimulated skin in the absence of concomitant VEGF. Surprisingly, there was no significant recruitment of CD45+, CD11b+, or F4/80+ myeloid, or mast cells in TetON-HIF-1:VEGF mice after 14 d of HIF-1 induction despite significant up-regulation of the VEGFR1 ligand, PlGF (Figs. 4F and 5 A–C and Fig. S4). By day 30, a significant increase in both CD45 and CD11b myeloid cells was achieved in TetON-HIF-1:VEGF mice; however, these myeloid cells were insufficient to facilitate neovascularization in the absence of epithelial VEGF (Fig. 5 A–C and Fig. S4). Myeloid cell infiltration after chronic HIF-1 gain of function in DOX day 30 TetON-HIF-1:VEGF mice was similar to our previous work with germ-line skin-targeted constitutive HIF-1 mutants in transgenic mice (37). There, stromal myeloid cell trafficking was regulated by chemokines not VEGF.
12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Inflammation Produces Extensive Vascular Remodeling with Minimal Microvascular Density Increase.
Topical phorbol ester induces angiogenesis via enhanced VEGF secretion from the activated epithelium (38, 39). Single-dose TPA treatment produces transient skin changes resolving after 3–4 d in wild type mice. Previously, we discovered that germ-line K14-HIF-1 transgenic mice responded to single-dose TPA challenge with a marked and prolonged stromal and intraepithelial neutrophil infiltrate persisting for 3 wk (37). Neutrophil recruitment was secondary to HIF-1–enhanced NFκB signaling and NFκB chemokine target gene up-regulation by transgenic keratinocytes. We capitalized on this strategy to determine whether the lack of HIF-1–mediated neovascularization in TetON-HIF-1:VEGF vasculature could be rescued. TetON-HIF-1:VEGF and TetON-HIF-1:VEGF were DOX-induced for 14 d to prime the keratinocytes and stroma and emulate germ-line constitutive activation. One dose of topical TPA was followed by ear harvest 10 d later. As reported, TPA treatment produced a marked recruitment and retention of CD45 stromal myeloid cells in TetON-HIF-1:VEGF mice (37), without affecting the frequency of CD11b macrophages (Fig. S5 A and B). Both TPA-treated DOX day 14 TetON-HIF-1:VEGF and TetON-HIF-1:VEGF mice ear epidermis was similarly punctuated by microabscesses filled with neutrophils (Fig. S6, Right), an observation consistent with our previous findings (37). Moreover, TPA did not further augment VEGF expression or neovascularization in TetON-HIF-1:VEGF mice (Fig. 6 A and B). In contrast, single-dose TPA in TetON-HIF-1:VEGF mice produced a twofold increase in VEGF expression associated with a similar fold increase in neovessel area. TPA also produced a similar influx of neutrophils in TetON-HIF-1:VEGF mice (Fig. S5A, Left and B, Upper). Partial rescue of HIF-1–mediated neovascularization in TetON-HIF-1:VEGF mice was associated with expansion of an epidermal population of unrecombined VEGF alleles and abscess-derived neutrophils (Fig. S5B). However, VEGF expression was undetectable in the majority of stromal neutrophils not associated with intraepidermal abcesses. TPA treatment also produced a statistically significant increase in capillary and total vessel volume, and a trend towards an increase in vessel tortuosity, as determined by OR-PAM (Fig. S7 A and B).
Mouse Development, Target Expression Analysis, Photoacoustic Microscopy, and Inflammation Challenge.
Detailed description of the mouse intercrosses to generate target and control genotypes, tissue molecular and expression analysis, photoacoustic microscopy, and inflammation challenge experiments are available in SI Materials and Methods. The Animal Studies Committee of Washington University in St. Louis approved all animal care and experimental procedures.
Statistical Analysis.
The data are reported as the mean ± SD. Data from DOX-treated TetON-HIF-1:VEGF or TetON-HIF-1VEGF mice were compared either with day 0 or NTG mice treated with DOX for the same interval by using the unpaired Student t test (GraphPad Prism 5). There were 3–6 mice per group unless otherwise indicated.
Supplementary Material
Author contributions: S.O., S.H., K.M., L.V.W., and J.M.A. designed research; S.O., S.H., J.K., A.S., and R.E.S. performed research; R.S. and K.M. contributed new reagents/analytic tools; S.O., S.H., J.K., J.Y., L.V.W., and J.M.A. analyzed data; and S.O., S.H., J.Y., L.V.W., and J.M.A. wrote the paper.
Abstract
Although our understanding of the molecular regulation of adult neovascularization has advanced tremendously, vascular-targeted therapies for tissue ischemia remain suboptimal. The master regulatory transcription factors of the hypoxia-inducible factor (HIF) family are attractive therapeutic targets because they coordinately up-regulate multiple genes controlling neovascularization. Here, we used an inducible model of epithelial HIF-1 activation, the TetON-HIF-1 mouse, to test the requirement for VEGF in HIF-1 mediated neovascularization. TetON-HIF-1, K14-Cre, and VEGF alleles were combined to create TetON-HIF-1:VEGF mice to activate HIF-1 and its target genes in adult basal keratinocytes in the absence of concomitant VEGF. HIF-1 induction failed to produce neovascularization in TetON-HIF-1:VEGF mice despite robust up-regulation of multiple proangiogenic HIF targets, including PlGF, adrenomedullin, angiogenin, and PAI-1. In contrast, endothelial sprouting was preserved, enhanced, and more persistent, consistent with marked reduction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic microscopy, which provides noninvasive, label-free, high resolution, and wide-field vascular imaging, revealed the absence of both capillary expansion and arteriovenous remodeling in serially imaged individual TetON-HIF-1:VEGF mice. Impaired TetON-HIF-1:VEGF neovascularization could be partially rescued by 12-O-tetradecanoylphorbol-13-acetate skin treatment. These data suggest that therapeutic angiogenesis for ischemic cardiovascular disease may require treatment with both HIF-1 and VEGF.
Neovascularization is crucial for solid tumor growth and metastatic spread, for wound healing, and for tissue preservation and recovery after ischemia. Although tremendous progress has been made in understanding the molecular circuits regulating neovascularization in both benign and malignant disease, our knowledge remains incomplete. Solid tumors evade angiogenesis inhibitors, and efficacious therapeutic angiogenesis for tissue ischemia remains a tantalizing prospect.
The hypoxia-inducible factors-1 and -2 (HIF-1 and HIF-2) are αβ heterodimeric proteins that make crucial contributions to neovascularization in wound healing, and benign or malignant diseases (1, 2). HIFs are master regulatory transcription factors with >100 known, and potentially hundreds more, target genes containing the core RCGTG enhancer sequence (3–6). A collection of HIF target genes coordinates neovascularization at several levels including endothelial cell proliferation, microvessel tone, vascular remodeling, and proangiogenic myeloid cell recruitment (7).
Numerous reports suggested that HIF produced a more robust neovasculature, with more normal structure and function, compared with single angiogenic factor overexpression (2, 8–10). However, the contribution of individual target genes encoding vascular functions to the collective HIF-1 neovascular phenotype is unknown. Here, we tested the hypothesis that VEGF was dispensable for HIF-mediated neovascularization. We used a unique conditional model of adult neovascularization in the skin, the TetON-HIF-1 transgenic mouse (11) that displays multistage neovascularization, and myeloid cell recruitment and retention in the absence of disease. TetON-HIF-1 mice were engineered for germ-line Cre-mediated VEGF deletion in the same basal keratinocytes targeted for conditional adult HIF-1α induction. VEGF deletion abrogated neovascularization despite coordinate up-regulation of multiple angiogenic growth factors. Surprisingly, endothelial sprouting was activated after HIF-1 induction; however, these sprouts were nonproductive for neovessel development. Despite robust PlGF, adrenomedullin, and PAI-1 induction, HIF-1 activation in the absence of VEGF failed to affect vascular remodeling, and myeloid cell recruitment was markedly impaired. Thus, we provide evidence that VEGF is required for HIF-1–mediated adult neovascularization, but that certain elements of the angiogenic response proceed without this factor.
Click here to view.Acknowledgments
We thank Hans Peter Gerber and Napoleon Ferrara for the gift of the VEGF mice, Rick Bruick for the gift of the TRE-HIF-1α plasmid for construction of the TRE-HIF-1α transgenic mice, and Adam Glick for the gift of the K5-rtTA transgenic mice. This work was supported by National Institutes of Health Grants R01-CA90722, R01 EB000712, R01 NS46214, R01 EB008085, and U54 CA136398, and the Beatrice Roe Urologic Cancer Fund.
Footnotes
Conflict of interest statement: L.V.W. has financial interest in Microphotoacoustics, Inc., and Endra, Inc., which, however, did not support this work. Other authors declare no competing financial interest.
*This Direct Submission article had a prearranged editor.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1101321108/-/DCSupplemental.
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