Myoglobin overexpression inhibits reperfusion in the ischemic mouse hindlimb through impaired angiogenesis but not arteriogenesis

Abstract

Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg(+)) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg(+) mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg(+) mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease.

Figure 1

Myoglobin overexpression impairs perfusion recovery after FAL. A: Laser Doppler perfusion recovery curve [ischemic ligated leg (L); normalized to nonischemic sham (R), leg] between myoglobin overexpressing transgenic mice (MbTg⁺) mice and WT littermate controls out to day 28 (n = 9 and 5, respectively). B: Early perfusion recovery within the first 7 days after FAL (n = 14 and 9, respectively). Functional recovery as determined by after FAL weight recovery (C) and foot use (D) [scaled from 0 (normal) to 3]. ^∗P < 0.05 between MbTg⁺ and WT within the given time point. ^†P < 0.05 between MbTg⁺ and WT recovery curves across all time points.

Figure 2

Collateral artery development in gracilis adductor muscle induced by FAL. A: Schematic illustration showing the position of the femoral artery ligation (X) relative to the gracilis collateral arteries (dashed-line boxes) that experience a significant increase in flow and undergo arteriogenesis. Flow directions are denoted with white arrows. B: Gracilis muscle whole mount regions from the ligated and unligated limbs of WT and myoglobin overexpressing transgenic mice (MbTg⁺) mice 28 days after FAL fluorescently labeled for smooth muscle α-actin to identify and quantify collateral artery remodeling (arrows). Images were taken from the collateral artery regions shown in A. C: Whole mount collateral artery diameters were quantified, showing outward remodeling of collateral arteries within the ligated limb starting at day 7 after FAL, but no additional growth by day 28 after FAL. There were no differences between MbTg⁺ and WT mice (n = 5 and 4 at day 7 and n = 11 and 7 at day 28, respectively). Scale bars: 500 μm (A–C). ^∗P < 0.05 between ligated versus unligated limbs within MbTg⁺ and WT mice.

Figure 3

Morphometric muscle and capillary analysis within distal calf muscle cross sections. Endothelial cells were labeled with CD31 and imaged in the oxidative (plantaris and deep gastrocnemius) and glycolytic (superficial gastrocnemius) regions of the calf muscle (Supplemental Figure S2). A–F: Quantification of capillary to muscle fiber ratio (A and B), mean fiber cross-sectional area (C–D), and percentage of regenerating fibers (E–F) within the glycolytic and oxidative regions of ligated and unligated limbs in myoglobin overexpressing transgenic mice (MbTg⁺) and WT control mice at 7 days [n = 5 and 4, respectively (A, C, and E)] and at 28 days [n = 11 and 7, respectively (B, D, and F)] days after FAL. ^∗P < 0.05 between groups denoted by bars. ^†P < 0.05 between MbTg⁺ and WT within ligated or unligated muscles.

Figure 4

Myoglobin transgene production increased within ischemic calf muscle. A and B: Cross sections (low magnification, ×4) of immunolabeled (hemaglutinnin)-tagged myoglobin transgene production using anti-hemaglutinnin epitope antibody (green) in myoglobin overexpressing transgenic mice (MbTg⁺) (A), and WT mice (B). Autofluoresence (red) and nuclear counterstain (blue) provide contrast for muscle visualization. Yellow borders define the oxidative region (OX), containing the plantaris muscle (PL) and glycolytic regions (GL) of the calf muscle. C–F: Images of myoglobin transgene production in nonischemic [unligated (C and D)] and ischemic [ligated (E and F)] limbs in oxidative (C and E) and glycolytic (D and F) regions of the calf muscle (high magnification, ×20). Scale bars: 500 μm (A–F).