Critical limb-threatening ischaemia and microvascular transformation: clinical implications.

BACKGROUND AND AIMS: Clinical management of critical limb-threatening ischaemia (CLTI) is focused on prevention and treatment of atherosclerotic arterial occlusions. The role of microvascular pathology in disease progression is still largely unspecified and more importantly not utilized for treatment. The aim of this explorative study was to characterize the role of the microvasculature in CLTI pathology. METHODS: Clinical high-resolution imaging of CLTI patients (n = 50) and muscle samples from amputated CLTI limbs (n = 40) were used to describe microvascular pathology of CLTI at the level of resting muscle blood flow and microvascular structure, respectively. Furthermore, a chronic, low arterial driving pressure-simulating ischaemia model in rabbits (n = 24) was used together with adenoviral vascular endothelial growth factor A gene transfers to study the effect of microvascular alterations on muscle outcome. RESULTS: Resting microvascular blood flow was not depleted but displayed decreased capillary transit time (P < .01) in CLTI muscles. Critical limb-threatening ischaemia muscle microvasculature also exhibited capillary enlargement (P < .001) and further arterialization along worsening of myofibre atrophy and detaching of capillaries from myofibres. Furthermore, CLTI-like capillary transformation was shown to worsen calf muscle force production (P < .05) and tissue outcome (P < .01) under chronic ischaemia in rabbits and in healthy, normal rabbit muscle. CONCLUSIONS: These findings depict a progressive, hypoxia-driven transformation of the microvasculature in CLTI muscles, which pathologically alters blood flow dynamics and aggravates tissue damage under low arterial driving pressure. Hypoxia-driven capillary enlargement can be highly important for CLTI outcomes and should therefore be considered in further development of diagnostics and treatment of CLTI.

EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF.

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.

T2 , T1ρ and TRAFF4 detect early regenerative changes in mouse ischemic skeletal muscle.

The identification of areas with regenerative potential in ischemic tissues would allow the targeting of treatments supporting tissue recovery. The regeneration process involves the activation of several cellular and molecular responses which could be detected using magnetic resonance imaging (MRI). However, to date, magnetic resonance (MR) relaxation parameters have received little attention in the diagnosis and follow-up of limb ischemia. The purpose of this study was to evaluate the feasibility of different MRI relaxation and diffusion tensor imaging parameters in the detection of areas showing early signs of regeneration in ischemic mouse skeletal muscles. T2 and T1ρ relaxation time constants, together with TRAFFn , T1 and diffusion tensor imaging, were evaluated to differentiate areas of regeneration in a mouse hind limb ischemia model before and 0, 1, 4, 7, 14 and 30 days after ischemia. All the measured relaxation times were longer in the areas of early regeneration compared with normal muscle tissue. The relaxation times increased after ischemia in the ischemic muscles, reaching a maximum at 4-7 days after occlusion, coinciding with the appearance of early signs of regeneration. Fractional anisotropy decreased significantly (p < 0.05) on days 1-4, whereas mean diffusivity, λ1 and λ2 decreased later, starting at day 7 after ischemia compared with the pre-operational time point. The percentages of areas with different tissue morphologies were determined based on histological analysis of the ischemic muscle cross-sections, and correlations between the percentages obtained and different relaxation times were calculated. The highest correlation between relaxation times and histology was achieved with T2 , T1ρ and TRAFF4 (R2 = 0.96, R2 = 0.92 and R2 = 0.84, respectively, p < 0.01). Early regenerative changes were visible using T2 , T1ρ and TRAFF4 MR relaxation time constants in skeletal muscle after ischemia. These markers could potentially be used for the identification of targets for therapies supporting muscle regeneration after ischemic injury.

Capillary Dynamics Regulate Post-Ischemic Muscle Damage and Regeneration in Experimental Hindlimb Ischemia.

This study aimed to show the significance of capillary function in post-ischemic recovery from the perspective of physiological parameters, such as blood flow, hemoglobin oxygenation and tissue regeneration. Muscle-level microvascular alterations of blood flow and hemoglobin oxygenation, and post-ischemic myofiber and capillary responses were analyzed in aged, healthy C57Bl/6J mice (n = 48) and aged, hyperlipidemic LDLR-/-ApoB100/100 mice (n = 69) after the induction of acute hindlimb ischemia using contrast ultrasound, photoacoustic imaging and histological analyses, respectively. The capillary responses that led to successful post-ischemic muscle repair in C57Bl/6J mice included an early capillary dilation phase, preceding the return of arterial driving pressure, followed by an increase in capillary density that further supported satellite cell-induced muscle regeneration. Initial capillary enlargement was absent in the LDLR-/-ApoB100/100 mice with lifelong moderate hypercholesterolemia and led to an inability to recover arterial driving pressure, with a resulting increase in distal necrosis, chronic tissue damage and a delay in the overall recovery after ischemia. To conclude, this manuscript highlights, beyond arterial collateralization, the importance of the proper function of the capillary endothelium in post-ischemic recovery and displays how post-ischemic capillary dynamics associate beyond tissue blood flow to both hemoglobin oxygenation and tissue regeneration.

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules.

Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy.