**2. Pericytes and smooth muscle cells cause a stabilization of newly formed tumour vessels**

Endothelia cover the innermost cell layer of the blood vessels. This continuous endothelium is made impermeable for substances dissolved in the blood by the formation of tight junctions in a first approximation. The necessary exchange of substances between blood and tissues is tightly controlled by a highly selective transport mechanism [1]. The uncontrolled cell growth which prevails in tumours results in a relative disparity between the tumour tissue and the sufficient formation of vascular structures. The initiation of tumour angiogenesis is associated with a structural destabilization of existing blood vessels. This causes an abnormally increased vascular permeability, i.e., the existing endothelium is fenestrated, and endothelial cells lose contact with one another and the underlying basal lamina. Finally, contact with the surround‐ ing mural, peri-endothelial cells (pericytes for capillaries and SMC of larger blood vessels) is lost. This leads to the fact that the now mature and quiescent endothelial cells start to migrate and proliferate [2-4]. During angiogenesis, continuous endothelial cells (the particularly impermeable form of the endothelial cells) undergo phases where they are not continuous, so are discontinuous (angiogenic endothelial cells). Chemotactic stimuli and vascular active growth factors such as VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) secreted by tumour cells induce mobilization and migration of angiogenic endothelial cells towards the tumour cells, which then build up new small blood vessels [5, 6].

During tumour angiogenesis, the hierarchical order of the blood vessels in large, medium and small blood vessels that is found in normal vasculature is lost. New vessel formation in the

tumour is disordered in structure; chaotic vascular structures are formed with areas of apparent excess supply, in addition to areas with an undersupply of blood and in particular oxygen and nutrition. In addition, tumour vessels have peculiarities in their structure: tumour vessels run tortuously in the tissue, may end blindly (increased permeability of blood vessels), have arteriovenous shunts (shorting connections causing liquid transfer between normally separate vessels), or be directed opposite to the blood flow (heterogeneous perfusion of the tumour tissue). The endothelial lining is incomplete [4, 7, 8]. The newly formed vessel walls lack the smooth muscle elements in their walls, so that they cannot actively respond to physiological stimuli. For this reason, the newly formed angiogenic capillaries bear an increased risk of rupture. Because of these features, tumour vessels prove to be functionally inferior. This complicates the efficient administration of intravenous drugs in cancer therapy [9-12].

**1. Introduction**

28 Muscle Cell and Tissue

**tumour vessels**

This chapter provides a summary of the current literature addressing the importance of vascular wall-resident multipotent stem cells within the process of vascular remodelling. First, the role of pericytes and smooth muscle cells (SMC) causing stabilization of angiogenic tumour vessels will be discussed at the molecular and cellular level. This stabilization phase is crucial for the survival of newly formed vessels, as immature vessels may rapidly become subject to regression and cell death when the angiogenic stimulus is removed. The second part of the chapter will focus on vascular wall-resident multipotent stem cells and evaluate the contribu‐ tion of circulating progenitor cells versus vessel-resident stem cells in the generation of pericytes and SMC within the neovascularization process. Here, the hypothesis will be proved that tissue-resident multipotent stem cells which putatively reside within the vascular adventitia, rather than circulating multipotent stem cells, are the major source for pericytes and SMC in the vascular stabilization processes. Finally, the regulation of differentiation of

Aspects of vascular stabilization, e.g., some decisive factors for the mobilization of vesselresident stem cells and differentiation into pericytes and SMC, may have the potential for clinically relevant applications in themselves. A better understanding of the molecular

**2. Pericytes and smooth muscle cells cause a stabilization of newly formed**

Endothelia cover the innermost cell layer of the blood vessels. This continuous endothelium is made impermeable for substances dissolved in the blood by the formation of tight junctions in a first approximation. The necessary exchange of substances between blood and tissues is tightly controlled by a highly selective transport mechanism [1]. The uncontrolled cell growth which prevails in tumours results in a relative disparity between the tumour tissue and the sufficient formation of vascular structures. The initiation of tumour angiogenesis is associated with a structural destabilization of existing blood vessels. This causes an abnormally increased vascular permeability, i.e., the existing endothelium is fenestrated, and endothelial cells lose contact with one another and the underlying basal lamina. Finally, contact with the surround‐ ing mural, peri-endothelial cells (pericytes for capillaries and SMC of larger blood vessels) is lost. This leads to the fact that the now mature and quiescent endothelial cells start to migrate and proliferate [2-4]. During angiogenesis, continuous endothelial cells (the particularly impermeable form of the endothelial cells) undergo phases where they are not continuous, so are discontinuous (angiogenic endothelial cells). Chemotactic stimuli and vascular active growth factors such as VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) secreted by tumour cells induce mobilization and migration of angiogenic endothelial cells towards the tumour cells, which then build up new small blood vessels [5, 6]. During tumour angiogenesis, the hierarchical order of the blood vessels in large, medium and small blood vessels that is found in normal vasculature is lost. New vessel formation in the

vascular wall-resident multipotent stem cells into SMC will be discussed.

processes in these cells could lead to the identification of new therapeutic targets.

While angiogenesis describes new vessel formation by endothelial cells derived from preexisting vessels, postnatal vasculogenesis denotes vessel formation by assembly of endothelial and/or vascular progenitor cells [13, 14]. Thus, the active cellular component in these processes is granted by endothelial lineage cells, but neovascularization does not only depend on endothelial cell migration and proliferation with subsequent formation of endothelial tubes; it also requires pericyte coverage of vascular sprouts for vessel stabilization [15-18]. Thereby, the vascular network can mature by recruitment of pericytes as well as SMC to stabilize the immature tumour vessels (Figure 1).

**Figure 1. Ultrastructural analysis of angiogenic tumour vessels.** Subcutaneously grown B16F10 melanoma tumours were removed 28 days after tumour induction and subjected to electron microscopic analysis. The presence of fenes‐ trae (emphasized by arrowheads) in angiogenic endothelial cells (EC) corroborate the less mature and functional inferi‐ or phenotype of these tumour vessels. Upon vessel maturation, these fenestrae disappear (upper panel). Vascular remodelling can be further observed by association and integration of pericytes to the newly formed blood vessels, resulting in vascular stabilization and thus maturation of angiogenic endothelial cells (lower panel). At the structural level, the recruited pericytes are assembled into new capillaries and change cell morphology into a more flattened, smooth muscle cell-like phenotype. In some tumour vessels, vascular mural cells seem to be more regularly integrated into the wall of the new capillaries because of their tight contact with the endothelial cells, shown, for example, in their sharing the same basement membrane, thereby indicating vessel stabilization and maturation (arrows). On PC peri‐ cyte: SMC smooth muscle cell, TC tumour cell, Lu lumen. Scale bar 1µm upper panel, 5µm lower panel.

At the molecular level, for the expression of important signalling molecules or receptors, or cell adhesion molecules, there is a locally pronounced heterogeneity in the tumour vascular bed [19, 20]. For a long time, these findings were interpreted as if there were no restructuring processes (vascular remodelling) of newly formed blood vessels in terms of a re-stabilization in the tumour vascular bed. Recent findings, however, show that even tumour vessels undergo a reorganization in terms of vascular stabilization to a certain degree [21, 22]. Electron microscopy analyses indicate that partially stabilized blood vessels exist that differ in their architecture from the usual blood vessels next to structurally stabilized and mature blood vessels, so that angiogenic and less stabilized vessels are disordered and regarded as immature. In combination with the fact that tumours require blood vessels for progressive tumour growth, many new cancer therapies directed against the tumour vasculature (anti-vascular agents, anti-angiogenic agents) have been investigat‐ ed. It was thought that these anti-angiogenic therapies could destroy the tumour vascula‐ ture to deprive the tumour of oxygen and nutrients. By contrast, it was shown that the process of vascular remodelling in tumours was affected during treatment with angiogen‐ esis inhibitors [18, 23-26]. Besides a dramatic tumour regression observed some angiogen‐ esis inhibitors, the tumours also became resistant to prolonged anti-angiogenic therapy. The tumour regression was histological, revealing a reduction in tumour vascularity observed during treatment predominantly as a result of the loss of less mature and highly proliferative small-calibre vessels. The remaining vessels were characterized by an increase in vessel diameter, and by the association and integration of pericytes and SMC leading to vascular stabilization in terms of vessel maturation, and thus a normalization of the vascular. This finally leads to an alternative hypothesis, that certain anti-angiogenic agents can also transiently normalize the abnormal structure and function of tumour vascula‐ ture to make it more efficient for oxygen and drug delivery [26]. Meanwhile, an exten‐ sive arsenal of anti-angiogenic compounds is available, and their effectiveness is currently being tested in numerous clinical studies. Bevacizumab is a humanized monoclonal anti-VEGF antibody which neutralizes any VEGF isoforms and prevents the interaction of VEGF with the corresponding receptors [27-29]. Clinical trials with bevacizumab show synergis‐ tic anti-tumour and chemotherapeutic effects. The results of histological examination of tumour tissue in clinical trials with anti-angiogenic substances showed a stabilization of tumour vessels, which was associated with a reduction in vascular density in the tumour tissue [11, 30-32].

In general, bevacizumab is used as first-line drug in combination with conventional chemo‐ therapeutics in patients with metastatic colorectal cancer, unless contra-indicated. The continuation of bevacizumab beyond first-line progression is still controversial, due to a lack of prospective randomized evidence in this setting [33]. The clinical efficacy of angiogenesis inhibitors targeting vascular endothelial cells has not been as successful as initially hoped, and improved clinical outcomes have been observed in combination with chemotherapy or additional drugs for many types of human cancer. This may be at least partially due to the fact that anti-angiogenic therapy triggers vascular stabilization including pericyte coverage, and that pericyte coverage further impairs tumour vessel regression in response to anti-angiogenic treatment [34]. Furthermore, tumour vessels which are resistant to anti-angiogenic therapy are characterized by an increase in vessel diameter and a normalization of vascular structures.

At the molecular level, for the expression of important signalling molecules or receptors, or cell adhesion molecules, there is a locally pronounced heterogeneity in the tumour vascular bed [19, 20]. For a long time, these findings were interpreted as if there were no restructuring processes (vascular remodelling) of newly formed blood vessels in terms of a re-stabilization in the tumour vascular bed. Recent findings, however, show that even tumour vessels undergo a reorganization in terms of vascular stabilization to a certain degree [21, 22]. Electron microscopy analyses indicate that partially stabilized blood vessels exist that differ in their architecture from the usual blood vessels next to structurally stabilized and mature blood vessels, so that angiogenic and less stabilized vessels are disordered and regarded as immature. In combination with the fact that tumours require blood vessels for progressive tumour growth, many new cancer therapies directed against the tumour vasculature (anti-vascular agents, anti-angiogenic agents) have been investigat‐ ed. It was thought that these anti-angiogenic therapies could destroy the tumour vascula‐ ture to deprive the tumour of oxygen and nutrients. By contrast, it was shown that the process of vascular remodelling in tumours was affected during treatment with angiogen‐ esis inhibitors [18, 23-26]. Besides a dramatic tumour regression observed some angiogen‐ esis inhibitors, the tumours also became resistant to prolonged anti-angiogenic therapy. The tumour regression was histological, revealing a reduction in tumour vascularity observed during treatment predominantly as a result of the loss of less mature and highly proliferative small-calibre vessels. The remaining vessels were characterized by an increase in vessel diameter, and by the association and integration of pericytes and SMC leading to vascular stabilization in terms of vessel maturation, and thus a normalization of the vascular. This finally leads to an alternative hypothesis, that certain anti-angiogenic agents can also transiently normalize the abnormal structure and function of tumour vascula‐ ture to make it more efficient for oxygen and drug delivery [26]. Meanwhile, an exten‐ sive arsenal of anti-angiogenic compounds is available, and their effectiveness is currently being tested in numerous clinical studies. Bevacizumab is a humanized monoclonal anti-VEGF antibody which neutralizes any VEGF isoforms and prevents the interaction of VEGF with the corresponding receptors [27-29]. Clinical trials with bevacizumab show synergis‐ tic anti-tumour and chemotherapeutic effects. The results of histological examination of tumour tissue in clinical trials with anti-angiogenic substances showed a stabilization of tumour vessels, which was associated with a reduction in vascular density in the tumour

In general, bevacizumab is used as first-line drug in combination with conventional chemo‐ therapeutics in patients with metastatic colorectal cancer, unless contra-indicated. The continuation of bevacizumab beyond first-line progression is still controversial, due to a lack of prospective randomized evidence in this setting [33]. The clinical efficacy of angiogenesis inhibitors targeting vascular endothelial cells has not been as successful as initially hoped, and improved clinical outcomes have been observed in combination with chemotherapy or additional drugs for many types of human cancer. This may be at least partially due to the fact that anti-angiogenic therapy triggers vascular stabilization including pericyte coverage, and that pericyte coverage further impairs tumour vessel regression in response to anti-angiogenic treatment [34]. Furthermore, tumour vessels which are resistant to anti-angiogenic therapy are characterized by an increase in vessel diameter and a normalization of vascular structures.

tissue [11, 30-32].

30 Muscle Cell and Tissue

**Figure 2. Scheme of the extensive remodelling of the tumour vascular bed by a partial structural stabilization of blood vessels upon tumour progression.** Newly formed tumour vessels supply a highly dense network of immature and unstable vessels. Upon tumour progression, angiogenic vessels can mature by the association and integration of pericytes and smooth muscle cells preferentially in the tumour centre zone. This process is accelerated in tumour ther‐ apy when anti-angiogenic agents are applied. Further vessel maturation leads to vessels characterized by an increased diameter, a reduction of vascular density and mural cell integration, resulting in normalization of the vascular bed. This process is accompanied by an extensive necrosis of the neighbouring tumour areas while viable tumour cell rows are circularly arranged around stabilized large arteries.

This normalization is achieved by the recruitment and integration of mature pericytes in the vessel wall for capillaries as well as SMC for larger vessels (Figure 2). This process is accelerated in tumour therapy when agents that affect the formation of new vessels (anti-angiogenic agents) were applied [11]. In contrast, the presence of VEGF led to ablation of pericyte coverage on nascent vascular sprouts and vessel destabilization [35]. Thus, targeting pericyte recruit‐ ment, coverage and function in addition to endothelial cells may be suitable for promoting progress in anti-angiogenic tumour therapy [36, 37]. In addition, the use of angiogenesis inhibitors which lead to a normalization of tumour vessels in combination with conventional therapies such as radiation or chemotherapy should lead to enhanced efficacy of drug delivery and diminished toxicity [38-40].
