Conditioning of Monocytes in the Bone Marrow by Inflammatory Cytokines Inhibits Their Angiogenic Potential at Peripheral Sites of Ischemia
Authors: Gregory Alyssa D. , Capoccia Benjamin J. , Link Daniel C. 
Published: April 2020

There is considerable interest in the potential of cell-based approaches to mediate therapeutic angiogenesis for a number of acute and chronic vascular syndromes. Accumulating evidence suggests that monocytes play a key role in the regulation of angiogenesis at sites of ischemia. Our laboratory has recently demonstrated that the adoptive transfer of a small number of bone marrow monocytes significantly enhanced revascularization in a murine model of acute hindlimb ischemia (Capoccia, 2008). However, the number of donor monocytes recruited to sites of ischemia was small compared to recipient (endogenous) monocytes; only 1% of the total monocytes in the ischemic muscle were of donor origin. This observation suggested that the angiogenic capacity of endogenous monocytes in the ischemic recipient was impaired. To test this hypothesis, we harvested monocytes from the bone marrow of ischemic or control mice, adoptively transferred them into recipient mice 24 hours after surgical induction of hindlimb ischemia, and measured revascularization using laser Doppler imaging. Compared with control cells, monocytes harvested from ischemic mice had markedly reduced capacity to stimulate revascularization [ratio of blood flow in ischemic versus nonischemic limb: 0.92 ± 0.04 (control) and 0.60 ± 0.10 (ischemic) on day 14; p &lt; 0.05]. Interestingly, the ischemia-conditioned and control monocytes were recruited to the ischemic tissue equally. We next performed RNA expression profiling on monocytes harvested from the bone marrow of ischemic and control mice. The expression of a number of STAT3 target genes was induced, including SOCS3. Accordingly, using a phosphoflow assay, we observed a significant increase in STAT3 phosphorylation in ischemia-conditioned monocytes (mean fluorescent intensity: 34 ± 4) compared with control monocytes (17 ± 2; p&lt;0.05). Major activators of STAT3 in monocytes are granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6), two cytokines often elevated in inflammatory conditions. Indeed, the serum concentration of G-CSF and IL-6 was markedly increased in ischemic mice [850 ± 40 pg/mL (G-CSF); 1660 ± 540 pg/mL (IL-6)] compared with control mice [123±40 pg/mL (G-CSF); undetectable (IL-6) p&lt;.05 for both]. Interestingly, monocytes obtained from ischemic IL-6−/− or ischemic G-CSFR−/− mice and adoptively transferred into ischemic recipients were competent to accelerate revascularization in contrast to monocytes obtained from ischemic wild type controls. These observations demonstrate that loss of either G-CSF or IL-6 signaling is sufficient to restore the ability of ischemia-conditioned monocytes to enhance revascularization. Taken together these data provide evidence that the ischemic environment generates signals, including G-CSF and IL-6, which inhibit the angiogenic potential of monocytes that reside in the bone marrow. This “conditioning” of bone marrow monocytes provides a novel mechanism by which systemic cytokines regulate angiogenesis and tissue repair.