The Retinoid Agonist Tazarotene Promotes Angiogenesis and Wound Healing

Abstract

Therapeutic angiogenesis is a major goal of regenerative medicine, but no clinically approved small molecule exists that enhances new blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1,280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitro angiogenesis, promoting branching morphogenesis, and tubule remodeling. The proangiogenic phenotype is mediated by retinoic acid receptor but not retinoic X receptor activation, and is characterized by secretion of the proangiogenic factors hepatocyte growth factor, vascular endothelial growth factor, plasminogen activator, urokinase and placental growth factor, and reduced secretion of the antiangiogenic factor pentraxin-3 from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing.

Figure 1

Phenotypic screen for angiogenic activity of LOPAC ¹²⁸⁰ small molecule library. (a) Left: Screening workflow. Primary human umbilical vein endothelial cells (HUVEC) were seeded onto Matrigel coated 384-well plates and incubated with Library Of 1,280 Pharmacologically Active Compounds (LOPAC¹²⁸⁰) compounds at a concentration of 10 μmol/l. Right: Plate design. The position of negative controls (Vehicle dimethylsulfoxide (DMSO) alone, blue), positive controls (Suramin, 10 μmol/l, red) and LOPAC¹²⁸⁰ small molecules (green) is indicated. (b) Image analysis workflow: Top: Endothelial tubes were stained for F-actin using phalloidin and CellMask Green and then imaged using an Operetta high content microcopy system. The region-of-interest (ROI, middle panel) is created on 16-bit red channel images and is followed by automated image segmentation (bottom panel) to identify tubes (white mask), nodes (green), and branching points (red dots) using Metamorph image analysis software (Molecular Devices). Scale bar = 1 mm. (c) Left: Scatter-plot distribution showing the results of high content screening. The total tube length was normalized using the B-score method in WebCellHTS2. Inhibitors and enhancers (B-Score ≤−4 and ≥+2.6 respectively) are indicated below and above the orange lines. Negative controls (are shown in blue positive controls in red and LOPAC¹²⁸⁰ library molecules in green. Right: Representative microscopic images from negative (Vehicle alone) and positive (suramin) control wells, Scale bar = 400 µm.

Figure 2

Validation of active lead compounds. (a) Selected enhancer hits were retested manually using the endothelial tube formation assay in 96-well format. The tubes were imaged and quantified as described in Figure 1. Left: Representative images of active lead compounds at 10µmol/l. Scale bar = 500 µm. Right: the chemical structure, the molecular weight, the primary target of the compound and its effect on the total tube length (normalized to Vehicle 100%), n = 3 each concentration. Data are expressed as mean ± SEM, one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test, **P < 0.01; ***P < 0.001 compared with Vehicle. (b) The effect of active lead compounds was tested in an organotypic angiogenesis assay where human umbilical vein endothelial cells (HUVECs) are plated on to a confluent human dermal fibroblast (HDF) layer. The medium containing the compound (7µmol/l) was refreshed at 3 and 5 days following plating of endothelial cells. Cocultures were stained with an antibody against CD31 and imaged 7 days after endothelial cell plating. Nine fields were quantified for each well (n = 6 wells per group). Error bars, mean ± SEM, one-way ANOVA followed by Bonferroni's post hoc test, *P < 0.05; ***P < 0.001 compared with Vehicle. Scale bar = 1mm. (c) Dose-response curve showing the effect of Tazarotene on total tube length using the organotypic angiogenesis assay. Data are shown as mean ± SEM. The curve was fitted using Prism GraphPad, and EC₅₀ calculated as 0.8µmol/l. Scale bar = 200 µm. (d) High-power image of Vehicle and tazarotene treated endothelial tubes, demonstrating lateral filopodia protrusions (arrows). Scale bar = 50 µm (e) Effect of Tazarotene (3 µmol/l) on expression of FLT4 (VEGFR3) and KDR (VEGFR2) in cultured endothelial cells estimated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Error bars, mean ± SEM, *P = 0.02 compared with Vehicle, (n = 3 replicates derived from three independent experiments, analyzed using unpaired t-test).

Figure 3

Tazarotene induces angiogenesis through retinoic acid receptor (RAR) activation. Human umbilical vein endothelial cells (HUVEC) were seeded on to confluent human dermal fibroblast (HDF) cells. Medium containing the compound (3 µmol/l) was refreshed 3 days following plating of endothelial cells. Cocultures were stained with anti-CD31 and imaged 5 days after endothelial cell plating. Angiogenic phenotype parameters (total tube length, branching points, tube thickness, and total node area) of the formed network were measured using Metamorph image analysis software. Nine fields were quantified for each well as described in the methods. (a) Effect of natural retinoic acids (all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid), RAR isoform agonists (RARα (BMS753), RARβ (CD2314) and RARγ (BMS961)) and retinoic X receptor (RXR) agonist (SR 11237) on angiogenic phenotype, compared with the Tazarotene-induced phenotype (n = 3 wells per condition). Error bars, mean ± SEM, one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test, **P < 0.01; ***P < 0.001 compared with Vehicle. Scale bar = 200µm. Right: Heatmap of z-score scaled data from the four angiogenic parameters, followed by hierarchical clustering of compounds to create the dendrogram generated by Spotfire software. Compounds clustering together, exhibit similar phenotypic profiles. (b) Effect of the pan-RXR antagonist (UVI 3003), pan-RAR antagonist (BMS5493) and Tazarotene on angiogenic phenotype (n = 3 wells per conditions). Error bars, mean ± SEM, one-way ANOVA followed by Bonferroni's post hoc test, ***P < 0.001. Scale bar = 200 µm. Right: compound phenotypic profiling using hierarchical cluster analysis as described above. (c) Effect of Liarozole (3 µmol/l), a retinoic acid metabolism inhibitor, on angiogenic phenotype, n = 6 wells per conditions. Data are expressed as mean ± SEM. Unpaired-t-test, **P < 0.01; ***P < 0.001 compared with Vehicle. Scale bar = 200 µm. (d) RARRES1 mRNA abundance in Tazarotene-treated HUVEC and HDF assessed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) (n = 3 replicates derived from three independent experiments). Unpaired-t-test, **P < 0.01 compared with Vehicle. Reprinted from ref. with permission of organization/publisher.

Figure 4

Tazarotene enhances the proangiogenic paracrine effects of fibroblasts. (a) Left: representative images showing the effect of Tazarotene (7 µmol/l) on cell number and DNA synthesis in isolated human umbilical vein endothelial cells (HUVECs). HUVEC were cultured as single cells in presence of Tazarotene or Vehicle for 48 hours. EdU (10µmol/l) was added 6 hours before fixation. Cells were stained for EdU (green) and DAPI (blue). Scale bar = 200 µm. (b) Left: representative images showing the effect of Tazarotene (7 µmol/l) on cell number and DNA synthesis in HUVEC/ human dermal fibroblasts (HDF) coculture at presence of Tazarotene or Vehicle for 48 hours. Cells were stained for EdU (green) and CD31 (red). Scale bar = 200 µm. Right panels of a and b, quantitative data for total cell number and the proportion of EdU positive nuclei. Cell counts and EdU positive cells were normalized to the Vehicle control, which was set at 100%. Data from four wells for each concentration, and nine fields per well were analyzed, and are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001 compared with Vehicle. (c) Effect of 5µmol/l Tazarotene on angiogenesis-related secreted proteins from HDF cells using Proteome Profiler Array. The inset shows the raw images from the array membrane for Vehicle (top) and Tazarotene (below), with letters corresponding to those on the bar-graph. Fluorescence intensity of each dot was normalized to the Vehicle control and presented as fold change (y-axis) on the bar graph. (d) Effect of 3 µmol/l Tazarotene placental growth factor (PGF) and pentraxin-3 (PTX3) mRNA expression estimated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Error bars, mean ± SEM, n = 3. Unpaired-t-test, **P < 0.01 compared with Vehicle. (e) Effect of Tazarotene on secreted PGF and PTX3 assayed by enzyme-linked immunosorbent assay (ELISA). n = 3 per treatment condition, error bars, mean ± SEM. One-way ANOVA followed by Bonferroni's post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001 compared with Vehicle.

Figure 5

Tazarotene promotes functional neovascularization in Matrigel implants. Mice were injected subcutaneously with Matrigel supplemented with FGF-2 (250 ng/ml). Tazarotene (10 mg/kg) or Vehicle (dimethylsulfoxide (DMSO)) was injected intra-peritoneally (i.p.) daily for 6 days. (a) Representative photographs of implants from Vehicle control and Tazarotene treated mice. (b) Effect of Tazarotene on neovessel formation. Top: Histological sections of Matrigel implants double immunostained for CD31 (endothelial marker, green) and α-smooth muscle actin (α-SM actin, smooth muscle cell marker, red). The dotted line shows the edge of Matrigel implant. Scale bar = 120 µm. Bottom: Mature neovessel area (y-axis) in the Matrigel plug was quantified by measuring CD31-positive neovessel invested by α-smooth muscle actin-positive cells stained areas in three sections (with a 1-mm interval between sections) for each implant and divided by total implant area, n = 6 animals per treatment group, two implants per animal were studied. Data are presented as box plots, with maximum, minimum, and quartile range. Two-way analysis of variance (ANOVA) (****P < 0.0001) followed by Bonferroni's multiple comparisons test, *P < 0.05, ***P < 0.001 compared with Vehicle group. (c) Effect of Tazarotene on microvessel perfusion using microCT angiography. Top: microCT angiogram of Matrigel implants. Mice were infused with Microfil 12 days after Matrigel implantation and the harvested implant analyzed by 3D microCT imaging to visualize the microvasculature (orange). Middle: 3D-reconstruction of microCT images of Matrigel implants including the perfused microvessels: vessels inside Matrigel (green), vessels surrounding Matrigel (red), and the Matrigel implant (transparent gray). Bottom: The vessel volume inside the Matrigel was quantified using Amira image analysis software. Data are expressed as % (vessel volume/Matrigel implant volume) by box plots, with maximum, minimum, and quartile range, n = 6 mice, two implants per treatment group. Unpaired-t-test, *P < 0.05 compared with Vehicle group. Scale bar = 2 mm. (d) Effect of Tazarotene on microvessel perfusion using confocal microscopy. Representative Z-stack confocal images of thick sections of Matrigel implants (n = 3 mice, two implants per treatment). To visualize vessel perfusion, terminally anaesthetized mice were injected with biotinylated IsolectinB4 12 days after implantation, implants harvested and sections immunolabeled for CD31 (green, endothelial cells), α-SM actin (red, smooth muscle cells), DAPI (Blue, Nuclei), and for injected lectin by using streptavidin-Alexa 647 (purple, perfused vessels). The dotted line shows the edge of Matrigel implant. Scale bar = 120 µm.

Figure 6

Tazarotene promotes ear punch wound healing and neovascularization after injury. (a) Effect of Tazarotene on ear punch closure. Left: Representative images of ears from mice treated with Vehicle or Tazarotene. Tazarotene (10 mg/kg, i.p.) was administered daily for 6 days following the ear-punch. Wound size was measured after 12 days. Right: Quantification of the punch area (n = 6 animals per group). Data are presented as box plots, with maximum, minimum, and quartile range, unpaired-t-test, ***P < 0.001 compared with Vehicle group. (b) Left: Effect of Tazarotene on blood flow following ear-punch injury. Representative images showing blood flow measured in the wound area using speckle contrast laser imaging at 6 and 12 days after injury (n = 5 animals per group). The dotted circle denotes the region-of-interest. Right: Mean flow (flux) in the region-of interest was quantified using moorFLPI-2 software, and data expressed as mean ± SEM. Two-way analysis of variance (ANOVA) (**P < 0.01) followed by Bonferroni's multiple comparisons test, *P < 0.05 compared with Vehicle. (c) Effect of Tazarotene on wound histology at 14 days after injury. Left: Hematoxylin and eosin stained cross-sections with the 2-mm area of the original hole indicated. Arrows indicate the initial wound site identified by the cartilage cut. Middle: Enlargement of the boxed section in the previous panel. Arrow-heads indicate new hair follicles in the wound bed. Right: Picrosirius red stained cross-sections. Birefringence of picrosirius differentiates thick, mature collagen fibers (red/yellow) from thin, new collagen (green). Quantification of collagen density, data expressed as % (stained collagen area/regenerated area), n = 6 animals per group. Unpaired-t-test, *P < 0.05 compared with Vehicle. Dotted line shows the initial cut position. Scale bar 90 µm. (d) Effect of Tazarotene on cell proliferation in the wound area. Left: Representative confocal images of ear wound at 6 days after injury stained with upper panel, CD31 (green), α-SM actin (red), DAPI (blue) and EdU (white); lower panel, stained with EdU (green), Keratin 14 “K14” (red) and DAPI (blue). The arrow indicates the initial wound site. Scale bar = 90 µm. Right: EdU positive cells were quantified and normalized to the total cell number inside the regenerated area which is identified by the cartilage cut, n = 4 animals per treatment condition. Data are expressed as mean ± SEM. Unpaired-t-test, ***P < 0.001 compared with vehicle group. (e) Effect of Tazarotene on wound neovascularization at 14 days after injury. Left: Cross sections of ear punches were stained with CD31 (green), α-SM actin (red) and DAPI (nuclei). Arrows indicate the initial wound site. Scale bar = 90 µm. Right: CD31-positive microvessel area was quantified as a percentage of the regenerated area, n = 6 animals per group. Unpaired-t-test, ***P < 0.001 compared with Vehicle group (f,g) Effect of Tazarotene on new vessel formation in the regenerating zone. Z-stack confocal images of flat mounted ears at day 4 f and day 14 g after injury. The dotted line indicates the wound site, the arrows shows new hair follicles, and the asterisk indicates the ear hole. CD31 (green, endothelium) and α-SM actin (red, smooth muscle cell, and myofibroblasts). Insets in g show a global view of the wound at low magnification. Scale bar = 90 μm.

Figure 7

Tazarotene enhances excisional dorsal wound healing and wound bed vascularization. (a) Effect of Tazarotene on wound closure. Left: Representative images of skin from mice treated with Vehicle or Tazarotene. Scale bar = 5 mm. Right: Quantification of the wound closure (n = 6 animals per group). Data are presented as box plots, with maximum, minimum, and quartile range, unpaired-t-test, *P < 0.05 compared with Vehicle group. (b) Effect of Tazarotene on wound epithelization. Left: Hematoxylin and eosin stained cross-sections of wound center. Scale bar = 500 µmol/l. Arrows indicate the wound edges. Middle: Enlargement of the boxed section in the previous panel. Arrow-heads indicate new hair follicles in the wound bed. Scale bar = 500 µmol/l. Right: Cross sections of wound center were stained with Keratin 14 (green), α-SM actin (red) and DAPI (nuclei). Arrows indicate the initial wound site. Scale bar 90 μm. (c) Effect of Tazarotene on new vessel formation in wound bed. Left: Cross sections of wounds were stained with CD31 (green), α-SM actin (red) and DAPI (nuclei). Scale bar 50 μm. Right: CD31-positive microvessel area was quantified as a percentage of the wound bed area, n = 6 animals per group. Arrows indicate the wound edges. Unpaired-t-test, *P < 0.05 compared with Vehicle group.