Following their firm adhesion on vascular endothelium walls, LFA-1, VLA-4 and VLA-5 facilitates the transendothe- lial migration of CD34+ cells across the ECM-rich basal lamina and towards the SDF-1 gradient. VLA-4 which binds both fibronectin (FN) and VCAM-1, together with VLA-5 which binds to the RGD sequence of FN, are important in the extravasation of these cells through the ECM of endothelia and of the target organ. It has been shown that anti-LFA-1-blocking antibodies completely disrupted IL-8 induced mobilization of murine HSCs [20] and antibod- ies blocking VLA-4 significantly reduced the homing of human CD34+ cells into the bone marrow in fetal sheep [21]. Interestingly, a new proposed mechanism is that adhe- sion molecules may trigger signals that enhance both SDF-1 receptor, CXCR4, expression and functionality [22]. In view of this finding, it is likely that the interactions between the SDF-1 -- CXCR4 axis and adhesion molecules are highly complex, involving a two-way regulation rather than a simple upregulation of cell adhesion molecules by SDF-1 alone. 3.1.2 Role of SDF-1 in cell secretion As CD34+ cells migrate across the endothelium, they have to pass through the ECM-rich basal lamina membrane. Hence, MMPs are vital for enzymatic degradation of the ECM in this phase of the homing process. It has been shown that SDF-1 stimulates the secretion of MMP-2 and MMP-9 in CD34+ cells [7,23] and SDF-1-mediated cell migration is completely blocked by MMP-9 inhibitors [24]. Moreover, Barkho et al. have reported that MMP-3 was also secreted together with MMP-9 in response to SDF-1 treatment in neural progenitor cells [25]. These reports illustrate that there is interplay of MMPs with chemokines during cell migration in response to injury. In addition to MMPs, SDF-1 has been reported to induce secretion of VEGF in endothelial cells and lymphohematopoietic cells [18,26] and VEGF is a well known pro-angiogenic growth factor, implying that SDF-1 partici- pates in a variety of physiological processes not only on homing but also in angiogenesis. The role of SDF-1 in angiogenesis is discussed in detail in a later section of this review. CD34+ cell CD34+ cell Blood vessel Flow direction CD34+ cell SDF-1 Target organ Sialyated carbohydrate ligand PSGL-1 E-selectin P-selectin ICAM-1 VCAM-1 LFA-1 VLA-5 VLA-4 MMP-9 CXCR4 Legend Endothelial cells Basal lamina Fibronectin Figure 1. Simplified schematic diagram illustrating stem cell rolling interactions, SDF-1 interactions, and SDF-1 chemotaxis. CD34+/CXCR4+ cells rolling on E and P selectins that are expressed on endothelial cells. After rolling, CD34+/CXCR4+ cells are activated by SDF-1, which is secreted from target organ, and triggers LFA-1/ICAM-1 and VLA-4/VCAM-1 interactions to induce firm adhesion onto endothelial wall. Arrested CD34+/CXCR4+ stem cells will secrete MMP-9 to facilitate extravasation and migration through the underlying basal lamina ECM using VLA-4 and VLA-5 integrin receptors to FN. Finally, CD34+/CXCR4+ stem cells will reach the target organ via chemotaxis response to SDF-1. Modified from [19]. Lau & Wang Expert Opin. Biol. Ther. (2011) 11(2) 191 For personal use only.

4. Implication of SDF-1 in engineered tissue regeneration Current research has been focusing on employing SDF-1 in cell-based therapies for patients with cardiomyopathies, liver and neural injuries and also those after radiation treatment. In this section of the review, we discuss the different engi- neering approaches in which SDF-1 is used in setting up plat- forms for future cell-based therapies. Various strategies used in different cell recruitments and organ regenerations are summarized in Table 1. 4.1 Effect of SDF-1 on CD 34+ mesenchymal stem cell (MSC) homing SDF-1-induced chemotaxis has been well established in HSCs and much of the research in the past decades focuses on HSCs lineage-related tissue regeneration. In recent years, a small pool of MSCs was found to express the chemokine receptor, CXCR4, and response to SDF-1-induced homing and the expression decreases with increase number of passages [27-29]. Although the transcript for CXCR4 was strongly expressed, the surface expression of CXCR4 in MSCs was relatively low, suggesting that this receptor may be largely intracellularly expressed [28-30]. It is most likely that CXCR4 are continu- ously cycling to and from the cell surface membrane and endosomal vesicles via endocytosis unless stimulated by chemokines where they are mobilized to the surface [31]. Given the capability of MSCs to differentiate into various mesoderm lineages, this finding has sparked an enormous interest and at the same time opened new doors for SDF-1-related tissue engineering applications. Studies have shown that increased numbers of transplanted MSCs were homed to various target organs such as heart [32], brain [33] and pancreatic islets [34] with local injection of SDF-1. Furthermore, introduction of CXCR4 antagonist has shown to significantly inhibit the chemotaxis of MSCs toward SDF-1 [28], confirming that SDF-1--CXCR4 interaction regulates the migratory response in MSCs. As scaffolds are often used in tissue engineering, several groups have demonstrated that controlled release of SDF-1 from various biomaterials is able to activate MSCs homing. Kimura et al. have shown that local controlled release of SDF-1 from gelatin hydrogel implanted subcutaneously in mice results in stronger angiogenesis in the hydrogel com- pared with injection of SDF-1 solution [35]. Similarly, poly- caprolactone (PCL) [30], poly(lactic-co-glycolic acid) (PLGA) scaffolds [36] and poly (lactide ethylene oxide fumarate) hydro- gel [37] have also been used to achieve MSC recruitment. In particular, Schantz et al. have implantated an acellular PCL scaffolds together with microneedle apparatus into the subcu- taneous pocket in rat model. This set up has allowed the sequential delivery of VEGF, SDF-1 and bone morphogenetic protein-6 (BMP-6) in the in vivo environment. Results showed the presence of native MSCs infiltrating into the scaffold, with concomitant angiogenesis and vascularization. This elegant design has highlighted the importance of SDF-1 in stem cell homing but also the synergistic effect of various growth factors and chemokines in tissue engineering [30]. As mentioned earlier, the surface expression of CXCR4 on MSCs is low compared with HSCs. Several groups have attempted to increase the surface receptors and integrins on MSCs by gene transfection so as to enhance MSCs��� homing. Cheng and colleagues reported a greater than twofold increase in the number of MSCs homed to the site of infracted myocardium by CXCR4 overexpression via retroviral transduction of CXCR4 genes [38,39]. In addition, Thieme et al. demonstrated over expression of CXCR4 enhanced the chemotactic capacity of MSCs to invade collagen based scaffolds up to 800 ��m and 500 ��m in in vitro and in vivo environment respectively [40]. Similar findings were also reported where CXCR4 genes were overexpressed using adenovirus vector transfection [41]. Interestingly, MMP-9 was also found to be upregulated in CXCR4-overexpressing MSCs, probably to facilitate the migration of stem cells across the ECM-rich areas during homing [41]. 4.2 Localized release of SDF-1 improves angiogenesis and wound healing SDF-1-induced VEGF secretion in cells and CXCR4 is reported to profoundly modulate the angiogenic activity and homing capacity of endothelial progenitor cells (EPCs) [42,43]. Hence, much of the work was focused on using SDF-1 in the treatment of cardiomypathies and other ischemia-related diseases [43]. A report by Sasaki and colleagues has shown that SDF-1 facilitates vasculogenesis in infarcted myocar- dium and ischemic limbs. Any impairment in CXCR4 receptor signaling would reduce vascularization and disrupt restoration of blood flow to the ischemic tissues [44]. Further- more, pretreatment of EPCs with SDF-1 prior to transplan- tation upregulates the expression of cell surface a4 and aM integrin subunits, which are involved in cell homing to neo- vasculature, and enhances cell recruitment to site of neovascularization [45]. Tissue engineering scaffolds have also been employed to achieve local controlled release of SDF-1 in the form of an SDF-1-bound PEG fibrin patch for infarcted heart. A marked increase in ejection fraction and fractional shorten- ing was shown when measuring heart function after direct injection of SDF-1 or implantation of the SDF-1-bound PEG fibrin patch. However, direct injection could only induce early mobilization and migration of c-kit+ cells while long term migration of cells to infarct site up to 4 weeks could be achieved with the PEG fibrin patch [46]. Due to the short half-life of SDF-1 proteins in circulation, several research groups have employed genetic modification strategies to con- tinuously express SDF-1 in situ. Using both viral and non- viral gene transduction approaches, upregulation of SDF-1 expression was achieved either via direct transfection of SDF1 gene [47,48] or through transplantation of cells overexpressing Homing factor SDF-1 for regenerative medicine 192 Expert Opin. Biol. Ther. (2011) 11(2) For personal use only.