**3. Vascular wall-resident multipotent stem cells stabilize angiogenic tumour blood vessels by differentiation into pericytes and smooth muscle cells**

Until some years ago, the bone marrow and endothelial cell compartment lining the vessel lumen (sub-endothelial space) were thought to be the only sources providing vascular progenitor cells. Results published recently have identified the human vessel wall as a niche for stem cells [41-44]. Herein, the blood vessels themselves harbour progenitors and multipo‐ tent stem cells (vascular wall-resident EPCs, VW-EPCs and haematopoietic stem cells, HPCs), clearly indicating the presence of stem cell niches outside the bone marrow and the peripheral blood [45-48]. Arterial vessels have what is termed an adventitial regeneration zone, in which those various stem and progenitor cells reside (Figure 3). These cells are able to form vascular networks and are capable of differentiating into endothelial cells and CD68+ macrophages [43, 46, 49]. However, the blood vessel wall is made not only of endothelial but also peri-endothelial cells (pericytes/SMC) and adventitial cells. Thus, the adequate formation of new blood vessels under hypoxia, during ischaemia or in tumour neovascularization, depends on the presence and recruitment of these peri-endothelial in addition to endothelial cells. Accordingly, the stem cell niche "vasculogenic zone" also harbours mesenchymal stem cells (MSCs) [43, 46].

Pericytes play a central role in tumour angiogenesis and these cells significantly affect the success of anti-angiogenic therapies. Thus it is important to identify pericytes in different tumour entities [50, 51]. In capillaries, pericytes are in close contact with endothelial cells and share the same capillary basement membrane. Pericytes express alpha-smooth muscle actin (ACTA2) and thus they might have contractile properties. However, the origins of pericytes and of SMC in tumours, and the molecular mechanisms that govern their recruitment and association with tumour vessels, are not clear. Endothelial expression of the platelet-derived growth factor B (PDGF-B) was shown to trigger the recruitment of pericytes necessary for the remodelling of newly formed vessels in terms of vascular stabilization, so that immature vessels with or without pericytes are formed [52]. Using an 'in vitro angiogenesis' system, Nicosia and co-workers suggested that pericytes are formed by migration and de-differentia‐ tion of arterial SMC [53]. Interestingly, pericytes have been assumed to differentiate in situ from mesenchymal cells [54]. In line with the idea that pericytes might have their origin in MSCs, it has been shown that Sca-1-positive bone marrow (BM)-derived cells are recruited to the site of tumour progression using the RIP-Tag2 model of pancreatic cancer [55].

In line with these findings, several studies identified human vascular wall-resident CD44+ multipotent stem cells (VW-MPSCs) within the adult human vascular adventitia which were capable of differentiation into pericytes and SMC [45, 46, 56-59]. VW-MPSCs were shown to contribute to in vivo vessel morphogenesis by co-implantation of isolated VW-MPSCs and human umbilical cord vein endothelial cell (HUVEC) in a matrigel plug assay [46]. Within the plugs, implanted HUVEC formed blood perfused vessels. Co-implanted VW-MPSCs assem‐ bled at the new vessels and were differentiated into transgelin-positive/ACTA2-positive SMC/ pericytes, undoubtedly confirming that VW-MPSCs have the capability to differentiate into pericyte/SMC and thus contribute to morphogenesis of new vessels under in vivo conditions. Electron microscopic analysis further demonstrated at the ultrastructural level that VW-MPSCs were not only aligned to new capillaries but were also regularly integrated into the wall of new capillaries; for example, EC and pericytes are enclosed by the same basal lamina. Thus, a crucial hypothesis concerning the vessel-resident stem cells is that these cells are the "first-line" cells, which are available on the basis of their anatomic location as the first point of contact for tumour cells and for tumour cell-secreted factors [43, 46, 60, 61]. Moreover, it is hypothesized that MPSCs or smooth muscle progenitors, resident in the vessel wall, would serve as a source for local recruitment of cells to stabilize new immature vessels constructed only by endothelial cells. Under vascular restructuring processes (remodelling) these VW-MPSCs associate with the newly formed blood vessels of the tumour and differentiate into pericytes and SMC, which results in a stabilization of the newly formed vessels (Figure 3).

progenitor cells. Results published recently have identified the human vessel wall as a niche for stem cells [41-44]. Herein, the blood vessels themselves harbour progenitors and multipo‐ tent stem cells (vascular wall-resident EPCs, VW-EPCs and haematopoietic stem cells, HPCs), clearly indicating the presence of stem cell niches outside the bone marrow and the peripheral blood [45-48]. Arterial vessels have what is termed an adventitial regeneration zone, in which those various stem and progenitor cells reside (Figure 3). These cells are able to form vascular networks and are capable of differentiating into endothelial cells and CD68+ macrophages [43, 46, 49]. However, the blood vessel wall is made not only of endothelial but also peri-endothelial cells (pericytes/SMC) and adventitial cells. Thus, the adequate formation of new blood vessels under hypoxia, during ischaemia or in tumour neovascularization, depends on the presence and recruitment of these peri-endothelial in addition to endothelial cells. Accordingly, the stem cell niche "vasculogenic zone" also harbours mesenchymal stem cells (MSCs) [43, 46].

32 Muscle Cell and Tissue

Pericytes play a central role in tumour angiogenesis and these cells significantly affect the success of anti-angiogenic therapies. Thus it is important to identify pericytes in different tumour entities [50, 51]. In capillaries, pericytes are in close contact with endothelial cells and share the same capillary basement membrane. Pericytes express alpha-smooth muscle actin (ACTA2) and thus they might have contractile properties. However, the origins of pericytes and of SMC in tumours, and the molecular mechanisms that govern their recruitment and association with tumour vessels, are not clear. Endothelial expression of the platelet-derived growth factor B (PDGF-B) was shown to trigger the recruitment of pericytes necessary for the remodelling of newly formed vessels in terms of vascular stabilization, so that immature vessels with or without pericytes are formed [52]. Using an 'in vitro angiogenesis' system, Nicosia and co-workers suggested that pericytes are formed by migration and de-differentia‐ tion of arterial SMC [53]. Interestingly, pericytes have been assumed to differentiate in situ from mesenchymal cells [54]. In line with the idea that pericytes might have their origin in MSCs, it has been shown that Sca-1-positive bone marrow (BM)-derived cells are recruited to

the site of tumour progression using the RIP-Tag2 model of pancreatic cancer [55].

In line with these findings, several studies identified human vascular wall-resident CD44+ multipotent stem cells (VW-MPSCs) within the adult human vascular adventitia which were capable of differentiation into pericytes and SMC [45, 46, 56-59]. VW-MPSCs were shown to contribute to in vivo vessel morphogenesis by co-implantation of isolated VW-MPSCs and human umbilical cord vein endothelial cell (HUVEC) in a matrigel plug assay [46]. Within the plugs, implanted HUVEC formed blood perfused vessels. Co-implanted VW-MPSCs assem‐ bled at the new vessels and were differentiated into transgelin-positive/ACTA2-positive SMC/ pericytes, undoubtedly confirming that VW-MPSCs have the capability to differentiate into pericyte/SMC and thus contribute to morphogenesis of new vessels under in vivo conditions. Electron microscopic analysis further demonstrated at the ultrastructural level that VW-MPSCs were not only aligned to new capillaries but were also regularly integrated into the wall of new capillaries; for example, EC and pericytes are enclosed by the same basal lamina. Thus, a crucial hypothesis concerning the vessel-resident stem cells is that these cells are the "first-line" cells, which are available on the basis of their anatomic location as the first point of contact for tumour cells and for tumour cell-secreted factors [43, 46, 60, 61]. Moreover, it is hypothesized that MPSCs or smooth muscle progenitors, resident in the vessel wall, would serve as a source for local recruitment of cells to stabilize new immature vessels constructed

**Figure 3. Vascular remodelling.** New vessel formation by both angiogenesis and postnatal vasculogenesis is a prereq‐ uisite for tissue regeneration and several diseases including tumour progression and atherosclerosis. Vascular stabili‐ zation is achieved by the recruitment and integration of mature pericytes in the vessel wall for capillaries, as well as smooth muscle cells (SMC) for larger vessels. Intima, media (TM) and adventitia with vasa vasorum (VV) are fixed layers of the wall of large arteries and veins. The border between media and adventitia is marked by outer elastic membrane (green). The vasculogenic zone is a vascular mural zone located within the adventitia and close to the tuni‐ ca media which harbours different subsets of vascular wall stem cells. The central hypothesis concerning vascular wall-resident multipotent MSCs (VW-MPSC) is that these cells serve as major source for pericytes and SMC for stabili‐ zation of new vessels, or repair of pre-existing vessels under physiological conditions. Localized within the vascular adventitia, which serves as an interface between the inner parts of the vessel wall, including blood flow and the sur‐ rounding tissue, the VW-MPSCs might serve as an important therapeutic target. Under vascular restructuring process‐ es (remodelling), these MSCs associate with the newly formed blood vessels of the tumour and differentiate into pericytes and SMC, which results in a stabilization and thus normalization of angiogenic tumour blood vessels. VW-MPSCs' differentiation into SMC may be induced by tumours, inflammation and hypoxia in tissue areas around blood vessels, contributing to morphogenesis of new vessel walls (e.g., tumour vascularization, intimal lesions or neointima formation). In contrast to the direct action of MPSCs during tumour progression through becoming mobilized from their niche and subsequently differentiated at the site of injury, the protective effect of endogenous or exogenous ap‐ plied MSCs could also be related to the modulation of paracrine characteristics of these cells. HSP, haematopoietic stem cell; EPC, endothelial progenitor cell; PC, pericyte; yellow, endothelial cells; green basement membrane and elas‐ tic membrane; blue, SMC.

Multipotent MSCs were intensively analysed using in vitro studies: optimized conditions were identified for their expansion and potential for differentiation along mesodermal lineages, e.g., into bone, fat, muscle and cartilage [62-65]. A frequently used source of MSCs is the bone marrow [66]. Here, only 0.01 to 0.001 % of the mononuclear cells in the BM are MSCs. Fur‐ thermore, human MSCs (hMSCs) can be obtained from umbilical cord blood, placental blood, foetal liver and adipose tissue [67-71]. It is further hypothesized that so-called permanent tissue stem cells exist in virtually every tissue type [72, 73]. In view of the fact that the blood vessels' area is a common structure of all tissues and organs, it is obvious that vessel-resident stem and progenitor cells may have great potential in biomedicine [46, 59, 74-76]. Together with the fact that tissue-specific stem cells differentiate predominantly into the tissue type from which they derive, vessel-resident (multipotent) MSCs may be particularly well suited to contribute to the formation of new vessels.

In general, abnormal vasculature is a hallmark of solid tumours. The exact quantification of tumour vessels is useful to evaluate prognosis, because the degree of angiogenesis is associated with tumour aggressiveness and clinical outcome [77]. Together with the fact that pericytes and SMC play a central role in vascular remodelling of tumour vessels, their recruitment and stable integration into stabilized tumour vessels may determine the success of anti-angiogenic therapies [78, 79]. Accordingly, future therapies targeting both endothelium and pericytes may favour progress in anti-angiogenic treatment for malignant tumours [80]. Thus, it is important to identify the origin and localization of pericytes and SMC in tumour tissues from cancer patients to gain a better understanding of their role in tumour growth and metastasis as well as to improve the outcome of anticancer therapies. Concerning the hypothesis that multipotent stem cells of mesenchymal nature (MPSCs) which express the (neural) stem cell marker nestin are the major source for pericytes and SMC in vascular stabilization processes, nestin-GFP transgenic mice were used in order to track MPSCs' contribution to the vascular remodelling processes. Nestin-GFP transgenic mice express GFP under the regulatory elements of the nestin promoter [81]. For transgene construction, the second intron of nestin gene was utilized, which was known to drive the expression in neural stem and progenitor cells. Furthermore, the 5′ upstream region (promoter region) in the transgene construct was included, the regulatory function of which is still unclear [82, 83]. Thus, these mice were ideally suited for the evaluation of the role of nestin-positive cells during the vascular remodelling of tumour blood vessels (Figure 4). Besides this, the BM tissue-resident nestin-GFP-positive cells are localized in the wall of mouse aortas and express nestin while lacking CD34 expression. Using arterial slice cultures of ex vivo isolates, these cells can be mobilized from their niche by factors secreted from cultured tumour cell lines, and are capable of differentiating into pericytes and SMC. In line with these results, Lin et al. have shown that tissue-resident MPSCs isolated from different anatomic locations gain the capacity to modulate the formation of vasculature by tightly surrounding newly formed microvessels as perivascular cells using a matrigel plug assay [84]. Furthermore, it has been demonstrated that human MPSCs derived either from the vascular adventitia or the bone marrow efficiently stabilized nascent blood vessels in vitro by functioning as perivascular precursor cells [46, 85]. Furthermore, vascular wall-resident nestin-GFP-positive cells can be isolated and cultivated. Primary cell cultures exhibited typical MSC characteristics. According to the guidelines, clonally expanded cells adhered on plastic, differentiated into adipocytes, chondrocytes and osteocytes under certain cell culture condi‐ tions [86]. These findings are in line with previous reports. Recently it has been elegantly demonstrated that nestin-GFP-positive cells in the BM are enriched in mesenchymal stem cell activities and are pericyte-like [87].

Multipotent MSCs were intensively analysed using in vitro studies: optimized conditions were identified for their expansion and potential for differentiation along mesodermal lineages, e.g., into bone, fat, muscle and cartilage [62-65]. A frequently used source of MSCs is the bone marrow [66]. Here, only 0.01 to 0.001 % of the mononuclear cells in the BM are MSCs. Fur‐ thermore, human MSCs (hMSCs) can be obtained from umbilical cord blood, placental blood, foetal liver and adipose tissue [67-71]. It is further hypothesized that so-called permanent tissue stem cells exist in virtually every tissue type [72, 73]. In view of the fact that the blood vessels' area is a common structure of all tissues and organs, it is obvious that vessel-resident stem and progenitor cells may have great potential in biomedicine [46, 59, 74-76]. Together with the fact that tissue-specific stem cells differentiate predominantly into the tissue type from which they derive, vessel-resident (multipotent) MSCs may be particularly well suited to contribute to the

In general, abnormal vasculature is a hallmark of solid tumours. The exact quantification of tumour vessels is useful to evaluate prognosis, because the degree of angiogenesis is associated with tumour aggressiveness and clinical outcome [77]. Together with the fact that pericytes and SMC play a central role in vascular remodelling of tumour vessels, their recruitment and stable integration into stabilized tumour vessels may determine the success of anti-angiogenic therapies [78, 79]. Accordingly, future therapies targeting both endothelium and pericytes may favour progress in anti-angiogenic treatment for malignant tumours [80]. Thus, it is important to identify the origin and localization of pericytes and SMC in tumour tissues from cancer patients to gain a better understanding of their role in tumour growth and metastasis as well as to improve the outcome of anticancer therapies. Concerning the hypothesis that multipotent stem cells of mesenchymal nature (MPSCs) which express the (neural) stem cell marker nestin are the major source for pericytes and SMC in vascular stabilization processes, nestin-GFP transgenic mice were used in order to track MPSCs' contribution to the vascular remodelling processes. Nestin-GFP transgenic mice express GFP under the regulatory elements of the nestin promoter [81]. For transgene construction, the second intron of nestin gene was utilized, which was known to drive the expression in neural stem and progenitor cells. Furthermore, the 5′ upstream region (promoter region) in the transgene construct was included, the regulatory function of which is still unclear [82, 83]. Thus, these mice were ideally suited for the evaluation of the role of nestin-positive cells during the vascular remodelling of tumour blood vessels (Figure 4). Besides this, the BM tissue-resident nestin-GFP-positive cells are localized in the wall of mouse aortas and express nestin while lacking CD34 expression. Using arterial slice cultures of ex vivo isolates, these cells can be mobilized from their niche by factors secreted from cultured tumour cell lines, and are capable of differentiating into pericytes and SMC. In line with these results, Lin et al. have shown that tissue-resident MPSCs isolated from different anatomic locations gain the capacity to modulate the formation of vasculature by tightly surrounding newly formed microvessels as perivascular cells using a matrigel plug assay [84]. Furthermore, it has been demonstrated that human MPSCs derived either from the vascular adventitia or the bone marrow efficiently stabilized nascent blood vessels in vitro by functioning as perivascular precursor cells [46, 85]. Furthermore, vascular wall-resident nestin-GFP-positive cells can be isolated and cultivated. Primary cell cultures exhibited typical MSC characteristics. According to the guidelines, clonally expanded cells adhered on plastic,

formation of new vessels.

34 Muscle Cell and Tissue

**Figure 4. Nestin-GFP(+) multipotent cells are localized in the vasculogenic zone of murine aorta.** (A) Immunohisto‐ logical analysis of stem cell antigen-1 (Sca-1) expression in mouse aorta sections. Scale bar 100µm. (B) Immunofluores‐ cence analysis of nestin-GFP-positive MSCs in their native niche was performed using double immunostainings on mouse aorta sections combining antibodies against GFP (green) and SMA or CD34 (red). Dotted line marks the border between media and adventitia of the aortic wall. Scale bar 20µm. (C) Electron microscopic analysis indicates the pres‐ ence of undifferentiated cells (putative stem cells (pSC) in the vasculogenic zone (Ad) of the adventitia. eEM external elastic membrane, SMC smooth muscle cell, TM, tunica media, Coll collagen.

In order to determine the contribution of the tissue-resident MPSCs to the formation of tumour neovasculature, BM transplantation experiments were performed. Tissue-derived cells were tracked when wild-type BM cells were isolated from C57BL/6 mice and transplanted into lethally irradiated, age-matched, syngeneic, nestin-GFP transgenic recipients [81]. Tumours grown in reconstituted nestin-GFP transgenic mice which received wild-type BM showed that ACTA2-positive pericytes exclusively expressed GFP, demonstrating that nestin-GFP-positive pericytes derived from tissue-resident cells and not circulating (BM-derived) MSCs stabilize angiogenic vessels in tumours grown in those mice. In combination with intensive immuno‐ fluorescence analysis, these results strongly confirmed the hypothesis that nestin-GFP-positive MSCs are apparently involved directly in vascular remodelling processes in terms of vascular stabilization, serving as a major source for pericytes and SMC. Thus, vascular wall-resident MSCs have to be considered in future strategies for anti-angiogenic tumour therapy. According to this idea, nestin expression of human colorectal adenocarcinoma metastases under clinical treatment with bevacizumab showed a prominent stabilization of tumour vessels by increased integration of nestin-positive pericytes and/or SMC into the vessel wall [81]. Mature vessels from the tumour's surrounding area or healthy tissue, by contrast, down-regulated nestin expression. Nestin expression had already been considered to be specific for developing vascular smooth muscle cells (VSMC), whereas differentiated, postmitotic VSMC were negative for nestin [88]. Conclusively, nestin-targeted therapy may suppress tumour prolif‐ eration via inhibition of neovascularization and vessel stabilization in numerous malignancies, including colorectal cancer and melanomas. Nestin, an intermediate filament protein, is reportedly expressed in repair processes, various neoplasms, and proliferating vascular endothelial cells [89, 90]. It was recently reported to be expressed in proliferating endothelial progenitor cells, but not in mature endothelial cells. Tumour endothelium-specific expression is thought to depend on the first intron of the nestin gene, whereas neural stem cell-specific and thus MSC-specific expression is usually regulated by the second intron [90]. Therefore, expression of nestin was described to be relatively limited in proliferating vascular endothelial cells and EPCs. Using another but similarly constructed nestin-GFP plasmid generated nestin-GFP transgenic mouse, nestin-positive pericytes have been identified as the progenitors of all Leydig cell phenotypes, indicating that vascular cell types, acting like adult stem cells, play a critical role in organ formation [91]. Thus, these findings confirm the idea that addressing pericytes, in particular by nestin-targeted therapy, may be suitable to selectively address newly formed and partially stabilized tumour blood vessels.

From the literature, it appears to still be controversial whether and to what extent BMderived vascular progenitor cells or tissue-resident stem and progenitor cells contribute to neovascularization processes. BM-derived endothelial progenitor cells (BM-EPCs) have been shown to represent an alternative source of endothelial cells for adult neovascularization in the process defined as postnatal vasculogenesis [92, 93]. Thus, BM-EPCs might consti‐ tute a new and promising target for pro- or anti-angiogenic treatment strategies [94]. However, there is extensive variation about their contribution to tumour neovasculariza‐ tion of primary tumours, and the respective values range from 50 % incorporated BM-EPCs to undetectable numbers, demonstrating that the exact role of these cells in postnatal vasculogenesis is not quite clear [95-97]. These contradictory results may be due to the methodological difficulties in distinguishing BM-derived cells from intimately associated cells [94]. Furthermore, the effects of MSCs on tumour growth are still controversial. Interactions between MSCs and tumour cells might play an important role in tumour growth [98, 99]. Herein, MSCs have been shown to transmit their tumour-promoting activity via a paracrine mechanism of action: conditioned media derived from cultured BM-MSCs induced the expression of VEGF in tumour cells as well as the activation RhoA-GTPase and ERK1/2 [100]. Furthermore, BM-derived MSCs (also called mesenchymal stromal cells) have been reported to migrate to the site of tumour progression and to subsequently differentiate into carcinoma-associated fibroblast (CAF)-like cells, thereby representing tumour-promoting stromal cells. As CAFs express platelet-derived growth factor receptor (PDGFR) at a high level, a blockade of PDGF signalling pathways by imatinib treatment influenced the interaction between BM-derived MSCs and tumour cells in the tumour microenvironment and, hence, inhibited the progressive growth of colon cancer [101].

stabilization, serving as a major source for pericytes and SMC. Thus, vascular wall-resident MSCs have to be considered in future strategies for anti-angiogenic tumour therapy. According to this idea, nestin expression of human colorectal adenocarcinoma metastases under clinical treatment with bevacizumab showed a prominent stabilization of tumour vessels by increased integration of nestin-positive pericytes and/or SMC into the vessel wall [81]. Mature vessels from the tumour's surrounding area or healthy tissue, by contrast, down-regulated nestin expression. Nestin expression had already been considered to be specific for developing vascular smooth muscle cells (VSMC), whereas differentiated, postmitotic VSMC were negative for nestin [88]. Conclusively, nestin-targeted therapy may suppress tumour prolif‐ eration via inhibition of neovascularization and vessel stabilization in numerous malignancies, including colorectal cancer and melanomas. Nestin, an intermediate filament protein, is reportedly expressed in repair processes, various neoplasms, and proliferating vascular endothelial cells [89, 90]. It was recently reported to be expressed in proliferating endothelial progenitor cells, but not in mature endothelial cells. Tumour endothelium-specific expression is thought to depend on the first intron of the nestin gene, whereas neural stem cell-specific and thus MSC-specific expression is usually regulated by the second intron [90]. Therefore, expression of nestin was described to be relatively limited in proliferating vascular endothelial cells and EPCs. Using another but similarly constructed nestin-GFP plasmid generated nestin-GFP transgenic mouse, nestin-positive pericytes have been identified as the progenitors of all Leydig cell phenotypes, indicating that vascular cell types, acting like adult stem cells, play a critical role in organ formation [91]. Thus, these findings confirm the idea that addressing pericytes, in particular by nestin-targeted therapy, may be suitable to selectively address newly

From the literature, it appears to still be controversial whether and to what extent BMderived vascular progenitor cells or tissue-resident stem and progenitor cells contribute to neovascularization processes. BM-derived endothelial progenitor cells (BM-EPCs) have been shown to represent an alternative source of endothelial cells for adult neovascularization in the process defined as postnatal vasculogenesis [92, 93]. Thus, BM-EPCs might consti‐ tute a new and promising target for pro- or anti-angiogenic treatment strategies [94]. However, there is extensive variation about their contribution to tumour neovasculariza‐ tion of primary tumours, and the respective values range from 50 % incorporated BM-EPCs to undetectable numbers, demonstrating that the exact role of these cells in postnatal vasculogenesis is not quite clear [95-97]. These contradictory results may be due to the methodological difficulties in distinguishing BM-derived cells from intimately associated cells [94]. Furthermore, the effects of MSCs on tumour growth are still controversial. Interactions between MSCs and tumour cells might play an important role in tumour growth [98, 99]. Herein, MSCs have been shown to transmit their tumour-promoting activity via a paracrine mechanism of action: conditioned media derived from cultured BM-MSCs induced the expression of VEGF in tumour cells as well as the activation RhoA-GTPase and ERK1/2 [100]. Furthermore, BM-derived MSCs (also called mesenchymal stromal cells) have been reported to migrate to the site of tumour progression and to subsequently differentiate into carcinoma-associated fibroblast (CAF)-like cells, thereby representing tumour-promoting stromal cells. As CAFs express platelet-derived growth factor receptor

formed and partially stabilized tumour blood vessels.

36 Muscle Cell and Tissue

In general, considerable evidence is accumulating for the involvement of tissue-resident and in particular vessel-associated MPSCs in regenerative and pathological adult neovasculariza‐ tion [43, 102, 103]. In vitro experiments further suggested that proliferative SMCs are derived from the differentiation of multipotent vascular stem cell (MVSC) of the blood vessel wall instead of the de-differentiation of mature SMCs [104]. MVSCs-expressed markers including Sox17, Sox10 and S100β were cloneable, had telomerase activity, and differentiated into neural cells and mesenchymal stem cell (MSC)-like cells that subsequently differentiated into SMCs. In vivo experiments further demonstrated that MVSCs, rather than mature SMCs, repopulate the tunica media and form neointima after endothelial denudation injury [104]. Whether MVSCs were derived from the de-differentiation of mature SMCs was determined by lineage tracing using SM-MHC as a marker in SM-MHC-Cre/LoxP-enhanced green fluorescence protein (EGFP) mice [105, 106]. These studies support the hypothesis that vascular multipotent stem cells of a mesenchymal nature were activated and generated SMC by differentiation instead of a possible SMC de-differentiation of the vascular wall. We may conclude that, in addition to their above-described role in tumour vascularization, the aberrant activation and differentiation of vascular wall-resident multipotent stem cells may contribute the develop‐ ment of vascular diseases. These findings may have a transformative impact on vascular biology, vascular diseases and remodelling, and may lead to new therapies by using VW-MPSCs as a therapeutic target.
