Although reports show that adipogenesis and angiogenesis are tightly correlated during the growth of fat mass [3, 17, , –20], little is known about the mechanisms that regulate the extension of its vasculature. In the present study we report that hAT-derived CECs induce the chemotaxis of hAT-derived progenitor cells. Moreover, we demonstrate that the presence of hAT-derived CECs promotes the differentiation and organization of hAT-derived hAT-CD34+/CD31− progenitor cells into capillary-like structures via a SDF-1/CXCR-4-dependent pathway.
In the last years, several reports have contributed to the evidence that the SVF cells are phenotypically heterogeneous and can express under specific culture conditions adipogenic, osteogenic, chondrogenic, and myogenic lineage markers but also endothelial, epithelial, and neural markers [5, 8, 21, –23]. We have previously shown that native immunoselected CD34+/CD31− cells present in the hAT-derived SVF display angiogenic and adipogenic potentials. Indeed, this cell population is able to differentiate into adipocyte-like cells  and into endothelial-like cells in vitro and in vivo to promote the neovascularization of ischemic muscle in mice . Similar results were also described using adherent hAT-SVF [6, 7]. The mechanisms underlying such proangiogenic effects are still to be defined. Although the hAT-derived progenitor cells were shown to integrate into vessels in vivo [5, 6], they could also contribute to neovascularization through the production of proangiogenic factors [7, 24]. Nevertheless, such a population of progenitor cells may participate locally to the extension of the blood capillary network known to be associated with fat mass development. The present study was undertaken to analyze the potential crosstalk within the hAT between hAT-CD34+/CD31− progenitor cells and CECs. Indeed, the EC barrier has already been shown to interact with progenitor/stem cells [25, –27]. Human AT-derived CECs specifically stimulated the migration of hAT-derived CD34+/CD31− progenitor cells through the release of soluble proteins. Human AT-derived CECs induced a chemotactic response since no random motility was observed when the cells were incubated in the same compartment with endothelial cell-derived conditioned media. Moreover, since hAT-derived CD34+/CD31− progenitor cell chemotaxis was inhibited in the presence of pertussis toxin, which specifically prevents activation of Gi proteins coupled to chemokine receptors, the involvement of a chemokine ligand/receptor interaction was strongly suggested. Finally, when CXCR-4 from hAT-CD34+/CD31− progenitor cells was neutralized by antibodies  or by the CXCR-4 antagonist (AMD3100) , hAT-derived CD34+/CD31− progenitor cells failed to migrate in response to hAT-derived CEC conditioned medium. Since real-time PCR and immunocytochemistry analyses showed the expression of CXCR-4 in native hAT-derived CD34+/CD31− progenitor cells, the present results demonstrate that activation of CXCR-4 of the hAT-derived CD34+/CD31− progenitor cells is involved in the hAT-derived CEC-dependent chemotaxis. In agreement, earlier work reported immunoreactivity to CXCR-4 in hAT , and a recent publication showed that CXCR-4 overexpression in hAT-stromal cells regulated cell motility . The sole ligand of CXCR-4, described so far, is the CXCL12 chemokine also known as SDF-1 [32, –34]. SDF-1, initially described as a product of bone marrow stromal cells , is expressed by several tissues including dendritic cells, endothelial cells and pericytes from normal skin , osteoblasts, and ECs from the bone marrow [37, –39] and astrocytes and neurons from the brain . SDF-1 exists as two isoforms, α and β, originating from alternative splicing of the CXCL12 gene [41, –43]. Human AT-CECs expressed and released more SDF-1 than the other cell types present in hAT. Moreover, SDF-1 was detected in very low amounts in conditioned media from HUVECs that did not stimulate the CD34+/CD31− progenitor cell chemotaxis. Human AT-progenitor cells exhibited a concentration-dependent chemotaxis toward SDF-1α and SDF-1β that was inhibited in the presence of CXCR-4 antagonist or neutralizing antibodies. Taken together, the present findings suggest that SDF-1 released by hAT-CECs plays a major role in the CXCR-4-mediated chemotaxis of hAT-progenitor cells, although further experiments are needed to clearly rule out the involvement of other endothelial-derived factors. The couple SDF-1/CXCR-4 plays a pivotal role in multiple checkpoints of stem/progenitor cell biology in the bone marrow compartment including survival, proliferation, mobilization, and homing [10, 44, 45]. In a two-dimensional coculture system, hAT-derived progenitor cells were shown to incorporate into the network formed by hAT-CECs and to express the EC marker CD31, suggesting that hAT-CECs may, in addition to their effect on the migration, regulate the differentiation of hAT-CD34+/CD31−. To better define the role of paracrine factors, three-dimensional coculture experiments were performed. The results clearly showed that hAT-derived progenitor cells migrate and assemble into capillary-like structures only in the presence of hAT-CECs. Moreover, when CXCR-4 either was antagonized or neutralized, hAT-progenitor cells failed to appropriately migrate and assemble into tubular structures on matrigel substrate.
Taken together, the present results show that hAT-derived CECs, through their production of paracrine factors such as SDF-1, modulate the migration but also the differentiation and organization of the hAT-derived CD34+/CD31− progenitor cells through the activation of CXCR-4. CXCR-4 mRNA levels are known to be upregulated by hypoxia through HIF-1 activation via the hypoxia responsive element in the 5′ region of the CXCR4 gene [46, 47]. In the murine AT, hypoxic areas have been shown to be associated with obesity . Interestingly, the mRNA levels of CXCR-4 from the CD34+/CD31− were found to be positively correlated to HIF-1α transcripts of the CECs. A recent publication has reported that the recruitment of CXCR-4 positive progenitor cells to regenerating foci or tissues was mediated by hypoxic gradients via HIF-1 induced expression of CXCR-4/SDF-1 axis . Moreover, SDF-1 expression in ischemic tissue has been shown to primarily localize in ECs . It is thus tempting to speculate that hypoxia within the hAT may promote the migration of resident hAT-derived CD34+/CD31− progenitor cells at active sites of neovascularization in the fat mass.