Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease model

Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.

The role of lymphangiogenesis in inflammation has remained unclear. To investigate the role of lymphatic versus blood vasculature in chronic skin inflammation, we inhibited vascular endothelial growth factor (VEGF) receptor (VEGFR) signaling by function-blocking antibodies in the established keratin 14 (K14)-VEGF-A transgenic (Tg) mouse model of chronic cutaneous inflammation. Although treatment with an anti-VEGFR-2 antibody inhibited skin inflammation, epidermal hyperplasia, inflammatory infiltration, and angiogenesis, systemic inhibition of VEGFR-3, surprisingly, increased inflammatory edema formation and inflammatory cell accumulation despite inhibition of lymphangiogenesis. Importantly, chronic Tg delivery of the lymphangiogenic factor VEGF-C to the skin of K14-VEGF-A mice completely inhibited development of chronic skin inflammation, epidermal hyperplasia and abnormal differentiation, and accumulation of CD8 T cells. Similar results were found after Tg delivery of mouse VEGF-D that only activates VEGFR-3 but not VEGFR-2. Moreover, intracutaneous injection of recombinant VEGF-C156S, which only activates VEGFR-3, significantly reduced inflammation. Although lymphatic drainage was inhibited in chronic skin inflammation, it was enhanced by Tg VEGF-C delivery. Together, these results reveal an unanticipated active role of lymphatic vessels in controlling chronic inflammation. Stimulation of functional lymphangiogenesis via VEGFR-3, in addition to antiangiogenic therapy, might therefore serve as a novel strategy to treat chronic inflammatory disorders of the skin and possibly also other organs

Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo.

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models

Inhibition of CXCR4 reduces imiquimod-induced skin inflammation.

<p>(A, B) Real-time RT-PCR analysis of extracts of IMQ-inflamed ear skin (8 consecutive days) and non-inflamed skin (n = 5 per group). The expression of CXCR4 and SDF-1 was significantly upregulated in IMQ-treated ear skin compared to uninflamed skin. (C–E) Immunofluorescence stains of ear skin for CXCR4 revealed a significantly increased CXCR4⁺ tissue area in IMQ-treated mice as compared with uninflamed skin. (F) Mice (n = 5 per group) received AMD3100 or PBS injections every 12 hours. 12 hours after the first injection, IMQ was applied topically, followed by daily applications for 8 days. (G) Treatment with AMD3100 (△) significantly reduced ear swelling as compared with PBS-treated controls (□). (H–J) H&E stains of ear skin sections showed that AMD3100 treatment (I) significantly reduced epidermal thickening compared to PBS-treated mice (H). Scale bar represents 100 μm. (K) CXCR4 inhibition significantly reduced the number of intraepidermal BrdU⁺ proliferating cells in the inflamed ear skin, as compared with control mice. Data represent mean±SD. ^*P<0.05; ^**P<0.01; ^***P<0.001.</p

Inhibition of SDF-1 signaling alleviates chronic skin inflammation and inhibits leukocyte migration towards SDF.

<p>(A) On day -5, hemizygous K14 VEGF-A transgenic mice (n = 14) were sensitized with 2% oxazolone and challenged on day 0 with 1% oxazolone on the ears. Starting 7 days after challenge, mice received i.v injections of anti-SDF-1 antibody or isotype-matched IgG every second day. (B) Neutralization of SDF-1 (△) significantly reduced inflammatory ear swelling as compared with IgG-injected mice (□). Data represent mean ±SEM. (C-D) H&E stains of mouse ear sections at day 21 showed reduced edema and inflammatory cell infiltration in the anti-SDF-1 treated mice (D) in comparison to the IgG control group (C). One ear half is shown. Scale bar represents 100 μm. (E) In anti-SDF-1 treated mice, the weight of ear draining LNs was significantly reduced compared with controls. (F-H) Immunofluorescence staining for CD68 revealed a significant reduction in the percentage of area covered by macrophages in anti-SDF-1-treated animals as compared to IgG treatment. (I-L) Neutralization of SDF-1 decreased the size and numbers of blood vessels in the inflamed ear skin. (M) FACS analysis of CD11b⁺ splenocytes revealed a clear expression of CXCR4 on their surface. (N) SDF-1 promoted the chemotactic migration of CD11b⁺ splenocytes in vitro. (O-P) The chemotactic effect of SDF-1 was blocked by incubation with AMD3100 (O) or with an anti-SDF-1 antibody (P), but not with control IgG. Two independent experiments were performed. Data represent mean±SD. ^**P<0.01; ^***P<0.001.</p

Inflammatory angiogenesis is reduced by inhibition of CXCR4.

<p>(A–B) Representative fluorescent images of MECA-32⁺ blood vessels (red) and LYVE-1⁺ lymphatic vessels (green) in the inflamed ear skin of PBS (A) and AMD3100-treated (B) K14-VEGF-A transgenic mice. One ear half is shown. Scale bar represents 100 μm. (C) Quantitative image analysis of MECA-32⁺ blood vessels (BV) revealed significantly reduced numbers of blood vessels per millimeter basement membrane (BM) in the inflamed ear skin of AMD3100-treated mice, as compared with PBS-treated control mice. (D–E) FACS analysis of CXCR4 expression by dermal blood vascular endothelial cells (BEC; D) and lymphatic endothelial cells (LEC; E) revealed that CXCR4 is expressed by BEC but not by LEC in vitro. (F) Immunofluorescence staining for CXCR4 (red), von Willebrand factor (VwF, green) and Podoplanin (Podo, purple) of inflamed ear skin (confocal image) demonstrates specific CXCR4 expression by blood vessels (BV) but not lymphatic vessels (LV). Scale bar represents 20 μm. Data represent mean ±SD. ^*P<0.05.</p

Increased number of CXCR4⁺ cells in human psoriatic skin and up-regulation of CXCR4 and SDF-1 in experimental chronic skin inflammation.

<p>(A–B) Immunohistochemistry revealed, that in normal human skin, CXCR4 is mainly expressed in the epidermis. In psoriatic skin, CXCR4 is also expressed on a big number of infiltrating cells. Quantitative image analysis showed that significantly more CXCR4⁺ cells were present in the dermis of psoriatic skin lesions (n = 8) than in normal human skin (n = 8). Staining with isotype matched control IgG confirmed the specificity of the CXCR4 staining. Scale bar represents 100 μm. ^***P<0.001. (C) Real-time RT-PCR analysis of RNA obtained from whole ear skin extracts of inflamed K14-VEGF-A transgenic mice (day 21 after oxazolone challenge), untreated K14-VEGF-A transgenic, and wild-type mice (n = 5 per group). The expression of CXCR4 and SDF-1 was significantly up-regulated in uninflamed K14-VEGF-A transgenic mouse skin compared to wild-type mice and was further increased in the inflamed skin of K14-VEGF-A transgenic mice. The expression of CXCR7 was slightly lower in the skin of K14-VEGF-A transgenic mice. (D–E) Immunofluorescence stains for CXCR4 revealed that inflamed K14-VEGF-A transgenic mice have a significantly increased CXCR4⁺ tissue area as compared with uninflamed K14-VEGF-A transgenic mice and wild-type mice. Scale bar represents 100 μm. Data represent mean ± SD. ^*P<0.05; ^***P<0.001. ns, not significant.</p

Inhibition of CXCR4 reduces inflammatory cell infiltration into the skin and normalizes epidermal architecture.

<p>(A–B) H&E stains of ear skin sections at day 21 showed that AMD3100 treatment reduced edema formation, epidermal thickening and inflammatory cell infiltration. (C–D) CXCR4 inhibition reduced the number of intraepidermal BrdU⁺ proliferating cells in the inflamed ear skin. (E–H) The hyperproliferation-associated keratin 6 and loricrin, a marker of terminal epidermal differentiation, were less broadly expressed in the epidermis of AMD3100-treated mice than in PBS-treated mice. (I–L) Immunofluorescence staining of the two macrophage markers F4/80 and CD68 revealed a significant reduction in the percentage of area covered by macrophages in AMD3100-treated mice compared to PBS treatment. (M–N) Inhibition of CXCR4 decreased the number of intraepidermal CD8⁺ T-cells in the inflamed ear skin. One ear half is shown. (O–P) Computer-assesed quantification of epidermal thickness (O), number of intraepidermal BrdU⁺ cells (P), the percentage of covered area by F4/80 (Q) and CD68 (R) postitive macrophages and the number of intraepidermal CD8⁺ cells (S). Scale bars represent 100 μm. Data represent mean ±SD. ^*P<0.05; ^**P<0.01; ^***P<0.001.</p

Increased permeability of cutaneous lymphatic capillaries and enhanced blood flow in psoriatic plaques

Morphological abnormalities of microvessels are described in psoriasis. However, there are conflicting data as to whether their function is also altered.; Our aim was to study the morphology and function of the lymphatic capillaries of psoriatic skin.; Morphology and permeability of initial lymphatics were studied by microlymphography and densitometry in 20 patients. Perfusion was studied by laser Doppler fluxmetry.; Permeability of lymphatics in plaques was increased by 7.6% compared to unafflicted skin (p < 0.001). Lymphatic vessel density and the extension of dye in lymphatic networks were not significantly different between involved and uninvolved areas. Both sites showed a wide range of diameters of lymphatics. The median laser Doppler flux in plaques was increased by 144% (91-380%) compared to unaffected skin (p < 0.001).; Increased permeability of lymphatics and increased blood flow was demonstrated in vivo in psoriatic skin lesions. These findings may reflect the local inflammatory process and may be used as markers when studying new therapeutic approaches for psoriasis