Key Points Freshly isolated arterial/venous endothelial cells differ in their gene signature, which is only partially controlled by the Notch pathway. Eight transcription factors codetermine the arterial fingerprint in a complementary and overlapping fashion.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.

Indeed, while questions about safety, dose and administration route/timing/frequency are the first ones to be addressed when designing a stem-cell based clinical approach, there is accumulating evidence from recent (pre-)clinical studies that other issues may also be at stake. For instance, the choice of stem cells to be used and its precise mechanism of action, the need/possibility for concurrent tissue regeneration in case of irreversible tissue loss, the differentiation degree and specific vascular identity of the transplanted cells and the long-term survival of engrafted cells in the absence of a normal supportive tissue environment, should be well considered. Here, rather than presenting a comprehensive and extensive overview on the current literature on stem/progenitor cells and revascularization, we highlight some of the outstanding issues emerging from the recent (pre-)clinical literature that may co-determine the successful application of stem cells in a wide range of PVD patients in the future.

IntroductionThe vascular system is a bipolar complex network of arteries that transport oxygen-rich blood to all tissues and veins that bring oxygendeprived blood back to the heart. 1 Because of this bipolar set-up, arteries and veins feature anatomic and physiological differences. Unlike venous endothelium, arterial endothelium is surrounded by several layers of smooth muscle cells (SMCs), separated by elastic laminae, and embedded in a thick layer of fibrillar collagen. 2 Moreover, both vessel types have a differential susceptibility to atherosclerotic disease, possibly due to exposure to different levels of shear stress. Arterial and venous endothelial cells (ECs) also have a distinct molecular signature, and such molecular specification occurs before the onset of blood flow. 3 Indeed, arteriovenous (AV) specification of ECs is accomplished early in development and is associated with the expression of a specific complement of factors: venous endothelium is characterized by the expression of EphB4, 4 Lefty-1, 5 Lefty-2, 5 COUP-TFII, 6 and MYO1-␤, 5 and arterial ECs express high levels of Notch 1 and 4, 7 Dll-4, 8 EphrinB1 and EphrinB2, 4 Jagged-1 and -2, 7 connexin-40, and Hey-2 (gridlock zebrafish ortholog). 9,10 Studies in Xenopus, zebrafish, and mice have revealed that, besides blood flow, 11 vessel-intrinsic cues and-later in development-signals from outside the vasculature 12,13 are implicated in defining arterial or venous fate such as members of the TGF-␤ pathway, 14,15 VEGF isoforms,13,[16][17][18]17 angiopoietins, 19 the Notch pathway, 7,9,20-22 the patched pathway, 20 and COUP-TFII, a member of the orphan nuclear receptor superfamily. 6 Although it has been shown that some of these pathways are well conserved from zebrafish to mouse, less information is available on whether they have a similar role in humans. Because these molecular differences between arterial and venous ECs exist independently of blood flow and some of these factors work in an EC-intrinsic way, 2 it should be possible to manipulate some or all of these to endow ECs with an arterial or venous fate. Consistent with this notion, recent studies have suggested that arterial markers can be induced in primary mature ECs. 5,13,21,23,24 Many different stem cell populations, including bone marrow (BM) mononuclear cells, AC133 ϩ endothelial progenitor cells, and embryonic stem cells have the potential to differentiate in vitro and in vivo into mature and functional ECs. 4,[25][26][27][28] We have recently described another stem cell population, multipotent adult progenitor cells (MAPCs), that differentiates into most somatic cell types, including functional ECs, in vitro and in vivo. [29][30][31][32][33] The question of whether and how these stem cells can be coaxed into arterial or venous ECs has thus far not been addressed. In this study, we analyzed the in vitro and in vivo arterial and venous endothelial differentiation of human MAPCs (hMAPCs) and hAC133 ϩ cells. Materials and methodsAdditional and extended descriptions of methods are inc...