Objectives 1) Develop and validate laser speckle flowmetry (LSF) as a quantitative tool for individual microvessel hemodynamics in large networks. 2) Use LSF to determine if structural differences in the dorsal skinfold microcirculation (DSFWC) of C57BL/6 and BALB/c mice impart differential network hemodynamic responses to occlusion. Methods We compared LSF velocity measurements to known/measured velocities in vitro using capillary tube tissue phantoms and in vivo using mouse DSFWCs and cremaster muscles. Hemodynamic changes induced by feed arteriole occlusion were measured using LSF in DSFWCs implanted on C57BL/6 and BALB/c. Results In vitro, we found that the normalized speckle intensity (NSI) versus velocity linear relationship (R2≥0.97) did not vary with diameter or hematocrit and can be shifted to meet an expected operating range. In vivo, DSFWC and cremaster muscle preparations (R2=0.92 and 0.95, respectively) demonstrated similar linear relationships between NSI and centerline velocity. Stratification of arterioles into predicted collateral pathways revealed significant differences between C57BL/6 and BALB/c strains in response to feed arteriole occlusion. Conclusions These data demonstrate the applicability of LSF to intravital microscopy microcirculation preparations for determining both relative and absolute hemodynamics on a network-wide scale while maintaining the resolution of individual microvessels.

Objective To estimate the relative influence of input pressure and arteriole rarefaction on gastrocnemius muscle perfusion in patients with peripheral arterial disease (PAD) after exercise and/or percutaneous interventions. Methods A computational network model of the gastrocnemius muscle microcirculation was adapted to reflect rarefaction based on arteriolar density measurements from PAD patients, with and without exercise. A normalized input pressure was applied at the feeder artery to simulate both reduced and restored ankle-brachial index (ABI) in the PAD condition. Results In simulations of arteriolar rarefaction, resistance increased non-linearly with rarefaction, leading to a disproportionally large drop in perfusion. Additionally, perfusion was less sensitive to changes in input pressure as the degree of rarefaction increased. Reduced arteriolar density was observed in PAD patients and improved 33.8% after 3 months of exercise. In model simulations of PAD, ABI restoration yielded perfusion recovery to only 66% of baseline. When exercise training was simulated by reducing rarefaction, ABI restoration increased perfusion to 80% of baseline. Conclusion Microvascular resistance increases non-linearly with increasing arteriole rarefaction. Therefore, muscle perfusion becomes disproportionally less sensitive to ABI restoration as arteriole rarefaction increases. These results highlight the importance of restoring both microvascular structure and upstream input pressure in PAD therapy.

Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

We tested whether the myeloid specific deletion of focal adhesion kinase (FAKΔmyeloid), which regulates macrophage differentiation and motility, impairs arteriogenesis and perfusion restoration. Laser doppler perfusion imaging showed a significant increase in perfusion recovery in FAKΔmyeloid as compared to WT mice (n=6/5, p=0.011) after unilateral femoral artery ligation. Gracilis collateral arteries showed no differences in end‐state (day 28) or baseline (non‐ischemic limb) diameter between FAKΔmyeloid [88.4 ± 11.2 μm/42.1 ± 6.5 μm (ischemic/non‐ischemic)] and WT mice [92.4 ± 1.0 μm/40.4 ± 4.7 μm], and there were no differences in angiogenesis in the ischemic calf muscles (capillary:muscle fiber ratio of 2.42 ± 0.20/2.24 ± 0.25 ischemic/non‐ischemic for FAKΔmyeloid vs 2.36 ± 0.32/2.06 ± 0.17 for WT control soleus, 1.57 ± 0.32/1.41 ± 0.18 for FAKΔmyeloid vs. 1.71 ± 0.32/1.46 ± 0.22 for WT oxidative gastrocnemius, and 0.98 ± 0.24/0.77 ± 0.13 for FAKΔmyeloid vs. 0.75 ± 0.03/0.75 ± 0.07 for WT glycolytic gastrocnemius). In conclusion, FAK in myeloid cells does not regulate arteriogenesis or angiogenesis but may affect perfusion recovery through alternative means, such as improved muscle regeneration and/or decreased necrosis. Supported by NIH R21HL098632, AHA 10GRNT3490001, the UVA Cancer Center (CCSG P30 CA44579 and Patients and Friends Fund), and the Arnold and Mabel Beckman Foundation Scholars Program.