Age-associated induction of cell membrane CD47 limits basal and temperature-induced changes in cutaneous blood flow

Abstract

Objective: We tested the hypothesis that the matricellular protein thrombospondin-1 (TSP1), through binding to and activation of the cell receptor CD47, inhibits basal and thermal-mediated cutaneous blood flow.

Background: Abnormal and decreased cutaneous blood flow in response to temperature changes or vasoactive agents is a feature of cardiovascular disease and aging. The reasons for decreased cutaneous blood flow remain incompletely understood. Furthermore, a role for matricellular proteins in the regulation skin blood flow has never been proposed.

Methods: C57BL/6 wild type, TSP1-null, and CD47-null 12- and 72-week-old male mice underwent analysis of skin blood flow (SkBF) via laser Doppler in response to thermal stress and vasoactive challenge.

Results: Young and aged TSP1- and CD47-null mice displayed enhanced basal and thermal sensitive SkBF changes compared with age-matched wild type controls. Nitric oxide-mediated increases in SkBF were also greater in null mice. TSP1 and CD47 were expressed in skin from young wild type mice, and both were significantly upregulated in aged animals. Tissue 3',5'-cyclic guanosine monophosphate, a potent vasodilator, was greater in skin samples from null mice compared with wild type regardless of age. Finally, treating wild type animals with a CD47 monoclonal antibody that inhibits TSP1 activation of CD47 enhanced SkBF in both young and aged animals.

Conclusions: These results suggest that secreted TSP1, via its cognate receptor CD47, acutely modulates SkBF. These data further support therapeutically targeting CD47 to mitigate age-associated loss of SkBF and maximize wound healing.

Figure 1. Cutaneous TSP1 and CD47 protein and mRNA are increased with age

Biopsies of skin from the dorsum of 12 and 72 week old male C57BL/6 mice were collected, tissue lysates prepared, protein separated by SDS-PAGE and Western blotted for TSP1 and CD47 (A). Densitometry represents the mean ± SD of blots prepared from distinct tissue samples from individual mice (n = 4 12 week old mice and n = 5 72 week old mice). * = p < 0.05 compared to 12 week old animals. (B). q-PCR analysis of TSP1 and CD47 mRNA (C). Results normalized to HPRT are the mean ± SD of material prepared from animal cohorts described in A. * = p < 0.05 compared to 12 week old animals.

Figure 2. Basal and thermal-mediated SkBF is increased in the absence of TSP1 and CD47

Under general anesthesia 12 week old male wild type, TSP1- and CD47-null mice underwent SkBF analysis with core temperature maintained at 37°C (A). Typical images of real time laser Doppler analysis are presented for wild type, TSP1- and CD47-null mice. Results represent the mean ± SD of 6 animals of each strain. * and # = p < 0.05 compared to wild type. Under general anesthesia 12 week old male C57BL/6 wild type, TSP1- and CD47-null mice under went SkBF analysis with laser Doppler beginning at 37°C, followed by controlled elevation of core temperature by 0.5°C with SkBF determined at each new core temperature, proceeding to a maximum core temperature of 38°C (B). Typical images of real time laser Doppler analysis are presented for wild type, TSP1- and CD47-null mice at the indicated core temperatures. Results represent the mean ± SD of 6 mice of each strain. * = p < 0.05 compared to wild type. Under general anesthesia 12 week old male C57BL/6 wild type, TSP1- and CD47-null mice under went SkBF analysis with laser Doppler beginning at 38°C, followed by controlled lowering of core temperature by 0.5°C with SkBF determined at each new core temperature, proceeding to a core temperature of 32°C (C). Results represent the mean ± SD of 6 mice of each strain. * = p < 0.05 compared to wild type.

Figure 3. NO-mediated effects on SkBF are limited by TSP1 and CD47

Under general anesthesia 12 week old male C57BL/6 wild type, TSP1- and CD47-null mice underwent SkBF analysis with laser Doppler with core temperature maintained constant at 37°C. After a 30 minute stabilization interval basal SkBF was determined, and animals were challenged with either vehicle (normal saline) or the primary NO donor DEA-NO (100 nmol/g body weight via rectal installation) (A), or the eNOS activator acetylcholine (ACh, 0.08 μg/gram weight via intravenous injection; wild type and TSP1-null mice) (B) and SkBF determined. Results are the mean ± SD of 6 animals of each strain. * and ** = p < 0.05 compared to wild type.

Figure 4. Aged TSP1- and CD47-null mice maintain SkBF and NO signaling

Under general anesthesia 72 week old male wild type, TSP1- and CD47-null mice with core temperature maintained at 37°C underwent SkBF determination via laser Doppler. Results are the mean ± SD of 6 animals of each strain. * = p < 0.05 compared to wild type. Dorsal cutaneous skin samples from 12 week (B) or 72 week (C) old male wild type, TSP1- and CD47-null mice were harvested, snap frozen in liquid nitrogen, pulverized, homogenized in lysis buffer and assayed for tissue cGMP as via ELISA (Amersham, GE Healthcare). Results presented are the mean ± SD of 4 animals of each strain. * and # = p < 0.05 compared to wild type.

Figure 5. Therapeutic blockade of CD47 activation increases SkBF

12 week (A) or 72 week (B) old male wild type C57BL/6 mice were treated with a CD47 blocking antibody (clone 301, 40 μg delivered as 10 μL of a 4 mg/mL stock in 100 μL of PBS in injected in the skin) or an isotype IgG2α control antibody as we had previously published. Three hours later animals underwent general anesthesia with their core temperature maintained at 37°C for 30 minutes prior to assessment of SkBF via laser Doppler. Results presented are the mean ± SD of 4 animals in each treatment group. * = p < 0.05 compared to isotype control antibody treated and untreated.