Figure 3
Figure 3. Pericyte marker expression and microvessel stability after HIF-1 withdrawal. (A) Representative lectin-perfused whole mounts (left column) and L-PAM images (middle and right columns) during HIF-1 induction (day 0, day 14) followed by DOX withdrawal 14/3 (14 days ON/3 days OFF), 14/14 (14 days ON/14 days OFF). Neocapillaries that developed farthest from the hair follicles remain after withdrawal accounting for a persistent 30% increase in vascularity compared with DOX day 0 or NTG controls. L-PAM images are obtained from the same TetON-HIF1 transgenic mouse imaged over 28 days. L-PAM detects development of a perifollicular neocapillary unit (purple arrowhead) with its adjacent vascular supply arch (red arrowhead), capillary luminal diminution with “single file” RBCs on withdrawal day 3 (purple arrowhead), and involution with persistence of peripheral microvessels by withdrawal day 14 (right column). (B) Quantification of microvessel density (MECA32 immunofluorescence, in black) and volume (L-PAM, in purple) in TetON-HIF-1 on DOX day 14, day 30, and day 60, and after increasing intervals of DOX activation followed by commensurate periods of withdrawal, 14/14, 14/28, 30/30, and 60/60 (n = 1-5). (C) Induction of endothelial cell apoptosis (dual-positive, MECA32/caspase-3 cells, arrowheads right panel) subjacent to epithelial basal cell compartments of the hair follicle and interfollicular epidermis (dashed dotted square and enlarged inset, white arrow denotes the hair shaft) after one day of DOX withdrawal (lower panel). (D) Thin section dual-endothelial (MECA32) and pericyte marker immunofluorescence demonstrates coverage of TetON-HIF-1 DOX day 14 induced microvessels by desmin, PDGFRβ, and NG2. α-SMA expression was restricted to arteries (A) and veins (V) as delineated morphologically and by the SMA expression pattern. (E) Confocal immunofluorescence demonstrates extensive and intimate microvessel coverage by desmin-positive pericytes at all time points after DOX induction (arrowheads delineate endothelial tip cells also shown in detail in supplemental Figure 1B). TetON-HIF-1 data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; ***P < .001. Bars represent 100 μm (A,D), 200 μm (C), and 20 μm (E).

Pericyte marker expression and microvessel stability after HIF-1 withdrawal. (A) Representative lectin-perfused whole mounts (left column) and L-PAM images (middle and right columns) during HIF-1 induction (day 0, day 14) followed by DOX withdrawal 14/3 (14 days ON/3 days OFF), 14/14 (14 days ON/14 days OFF). Neocapillaries that developed farthest from the hair follicles remain after withdrawal accounting for a persistent 30% increase in vascularity compared with DOX day 0 or NTG controls. L-PAM images are obtained from the same TetON-HIF1 transgenic mouse imaged over 28 days. L-PAM detects development of a perifollicular neocapillary unit (purple arrowhead) with its adjacent vascular supply arch (red arrowhead), capillary luminal diminution with “single file” RBCs on withdrawal day 3 (purple arrowhead), and involution with persistence of peripheral microvessels by withdrawal day 14 (right column). (B) Quantification of microvessel density (MECA32 immunofluorescence, in black) and volume (L-PAM, in purple) in TetON-HIF-1 on DOX day 14, day 30, and day 60, and after increasing intervals of DOX activation followed by commensurate periods of withdrawal, 14/14, 14/28, 30/30, and 60/60 (n = 1-5). (C) Induction of endothelial cell apoptosis (dual-positive, MECA32/caspase-3 cells, arrowheads right panel) subjacent to epithelial basal cell compartments of the hair follicle and interfollicular epidermis (dashed dotted square and enlarged inset, white arrow denotes the hair shaft) after one day of DOX withdrawal (lower panel). (D) Thin section dual-endothelial (MECA32) and pericyte marker immunofluorescence demonstrates coverage of TetON-HIF-1 DOX day 14 induced microvessels by desmin, PDGFRβ, and NG2. α-SMA expression was restricted to arteries (A) and veins (V) as delineated morphologically and by the SMA expression pattern. (E) Confocal immunofluorescence demonstrates extensive and intimate microvessel coverage by desmin-positive pericytes at all time points after DOX induction (arrowheads delineate endothelial tip cells also shown in detail in supplemental Figure 1B). TetON-HIF-1 data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; ***P < .001. Bars represent 100 μm (A,D), 200 μm (C), and 20 μm (E).

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