Vascular Normalization as a Therapeutic Strategy for Malignant and Nonmalignant Disease

Abstract

Pathological angiogenesis—driven by an imbalance of pro- and antiangiogenic signaling—is a hallmark of many diseases, both malignant and benign. Unlike in the healthy adult in which angiogenesis is tightly regulated, such diseases are characterized by uncontrolled new vessel formation, resulting in a microvascular network characterized by vessel immaturity, with profound structural and functional abnormalities. The consequence of these abnormalities is further modification of the microenvironment, often serving to fuel disease progression and attenuate response to conventional therapies. In this article, we present the “vascular normalization” hypothesis, which states that antiangiogenic therapy, by restoring the balance between pro- and antiangiogenic signaling, can induce a more structurally and functionally normal vasculature in a variety of diseases. We present the preclinical and clinical evidence supporting this concept and discuss how it has contributed to successful treatment of both solid tumors and several benign conditions.

Uncontrolled angiogenesis is driven by an imbalance of pro- and antiangiogenic signals. The judicious use of antiangiogenic therapies (e.g., anti-VEGF treatments in cancer) results in vascular normalization.

The successful functioning of all tissues depends on the establishment of a hierarchically structured, mature vascular network. As such, the development of new blood vessels— angiogenesis—plays a critical role in healthy human development. Angiogenesis in the human is most vibrant during embryogenesis and is relatively suppressed in the adult. It is usually a tightly regulated process, triggered by specific molecular and mechanical stimuli to meet the needs of the host and suppressed again by antagonistic stimuli when these needs have been met.

In contrast to the healthy state, a number of human diseases show a dysregulated excess of new blood vessel formation. Solid tumors are the best characterized example, with seminal work first performed more than 70 years ago confirming the importance of an abundant blood supply for tumor growth (Ide et al. 1939; Algire and Chalkley 1945). Unlike physiological angiogenesis, blood vessel development in solid tumors is not tightly controlled, but rather occurs relentlessly (Dvorak 1986; Chung et al. 2010; Carmeliet and Jain 2011). Molecular stimuli within solid tumors (hypoxia, acidosis, oncogenic signaling, growth factors, sex hormones, and cytokines) all induce the formation of new vessels (Vogelstein and Kinzler 2004; Ferrara 2005; Carmeliet and Jain 2011). Although the main purpose of such stimuli is to ensure a rich vascular supply for continuing tumor growth, the unyielding drive for angiogenesis results in a vascular network that is highly abnormal when compared to the organized structure of vessel networks in normal tissues (Jain 2005a, 2008). This structurally abnormal network leads to aberrations in local blood flow, fluid dynamics, and oxygenation that in turn can augment tumor growth and metastatic potential while diminishing response to cytotoxic therapies (Jain 2001, 2005b). More recently, similar abnormalities in vessel structure and function have been reported in a number of nonmalignant diseases (Carmeliet and Jain 2011; Goel et al. 2011). In each of these examples, disease progression is influenced by abnormalities in the microvasculature and the resultant abnormal microenvironment.

The discovery of vascular endothelial growth factor (VEGF) as the principal driver of tumor angiogenesis (Senger et al. 1983; Leung et al. 1989) rapidly prompted the development of antiangiogenic drugs to treat cancer, designed to inhibit VEGF's activity and hence promote vascular regression and tumor starvation (Folkman 1971; Kim et al. 1993). This rationale was supported by early preclinical trials demonstrating growth delays in mouse models of solid cancers after treatment with anti-VEGF antibodies (Kim et al. 1993). Unfortunately, results using such agents in clinical trials have been disappointing, with antiangiogenic monotherapy generally failing to invoke significant response rates or prolongations of survival in solid tumor patients (Jain et al. 2006; Giantonio et al. 2007). Indeed, clinical data suggest that anti-VEGF therapy cannot induce sustained shrinkage in human tumors such as breast and colorectal cancer. Intriguingly, however, the addition of anti-VEGF therapy to systemic chemotherapy has often proven to be an effective strategy, with patient outcomes superior to chemotherapy alone (Hurwitz et al. 2004; Sandler et al. 2006; Miller et al. 2007; Saltz et al. 2008; Reck et al. 2009). This suggests that although antiangiogenic therapies may not “starve” tumors in patients, they do in some way enhance the activity of cytotoxics—an intuitively paradoxical observation given that the efficacy of chemotherapy depends on the presence of an adequate tumor blood supply to ensure drug delivery. The “vascular normalization” hypothesis is a potential resolution of this paradox.

In this article, we present the vascular normalization hypothesis, which we first introduced in 2001 (Jain 2001). This hypothesis posits that rather than obliterating vessels, the judicious use of antiangiogenic therapy prunes some vessels and reverts the grossly abnormal structure and function of the remaining vasculature toward a more normal state, abrogating its deleterious effects on the tumor microenvironment. We summarize the preclinical and clinical studies providing evidence in support of this hypothesis. In addition, we discuss the nature of vascular abnormalities in nonmalignant disease, and possible benefits from normalizing these. We also present recent findings that have shed light on the key molecular players regulating the switch between a “normal” and “abnormal” microvessel phenotype, furthering the mechanistic understanding of vascular normalization.

VASCULAR ABNORMALITIES IN SOLID TUMORS

Solid tumors should be considered as entire “organs” in themselves. Much more than a mass of proliferating cancer cells, a tumor is an assembly of cancer cells, a blood vessel network, lymphatic vessels, and a variety of other cells all of which contribute to the local microenvironment. Angiogenesis within solid tumors is driven largely by hypoxia. This hypoxia, a hallmark of the tumor microenvironment, leads directly to the production of proangiogenic factors such as VEGF via modulation of oxygen sensing molecules (Semenza 2010). In addition, VEGF production in tumors is also fuelled by acidosis, activation of cellular oncogenes that lie upstream of VEGF, inflammatory cytokines, growth factors, and protumorigenic sex hormones (Vogelstein and Kinzler 2004; Ferrara 2005; Carmeliet and Jain 2011).

The microenvironmental abundance of VEGF and other proangiogenic factors drives continual angiogenesis and the production of an abnormal blood vessel network (Jain 2005a; Nagy et al. 2009). Structurally, vessels are often dilated, weave a tortuous path, and show heterogeneity of distribution such that certain areas within a tumor are hypovascular and others hypervascular (Less et al. 1991, 1997; Yuan et al. 1996; Tong et al. 2004; Jain 2005b; Baish et al. 2011). At the cellular level, proangiogenic factors induce weakening of VE-Cadherin-mediated endothelial cell (EC) junctions and EC migration, altering vessel wall architecture (Hobbs et al. 1998; Hashizume et al. 2000). Similarly, the perivascular cells (PVCs, comprised of pericytes and vascular smooth muscle cells [VSMCs]) are often only loosely attached to ECs and are reduced in number (Morikawa et al. 2002; Abramsson et al. 2003; Jain 2003; Inai et al. 2004; Tong et al. 2004). A number of important molecules regulate pericyte–EC interaction, including platelet-derived growth factor-B (PDGF-B), Ang-1, transforming growth factor-β (TGF-β), and sphingosine-1-phosphate (S1P) in healthy tissues (Jain 2003; Jain and Booth 2003). In tumors, proangiogenic factors promote PVC-EC dissociation, however. For example, VEGF induces the formation of VEGFR-PDGFRβ complexes in pericytes, which impede PDGFRβ-mediated adherence of ECs to pericytes (Greenberg et al. 2008). Finally, the perivascular basement membrane (BM) is also structurally abnormal in tumors—excessively thin or absent in certain regions and abnormally thick in others (Inai et al. 2004; Tong et al. 2004; Winkler et al. 2004; Kamoun et al. 2009).

A direct consequence of these structural derangements is marked aberration of tumor vascular function. The haphazard and bizarre distribution of vessels leads to heterogeneous blood flow, sluggish in some regions and excessive in others (Jain 1988, 2005b; Kamoun et al. 2010). In addition, reduced PVC coverage, EC dissociation, and an excess of vesiculo-vaculor organelles (VVOs) results in marked tumor vessel permeability, with excess extravasation of fluid and protein into the extracellular compartment (Jain 2005b). This leakiness, together with a relative absence of functional intratumoral lymphatic vessels (Padera et al. 2004; Hagendoorn et al. 2006), leads to a marked increase in the tumor interstitial fluid pressure (IFP) to a level that equilibrates with intravascular pressure, which results in reduced transvascular flow (Boucher et al. 1990, 1991; Roh et al. 1991; Less et al. 1992; Leunig et al. 1992; Stohrer et al. 2000; Tong et al. 2004; Willett et al. 2004). Furthermore, the compressive forces applied by the proliferating mass of cancer cells can cause vascular compression and collapse (Padera et al. 2004). The net result is a heterogeneous blood supply, and resultant hypoxia and acidosis (Jain 2005b).

The physiological changes described have a direct effect on solid tumor behavior. Hypoxic tumor cells often show a more aggressive phenotype, activating oncogenes and passing through an “epithelial to mesenchymal transition” (EMT), which heightens their metastatic potential (DeClerck and Elble 2010). Moreover, the hostile microenvironment impairs the function of antitumor immune cells, the delivery of which into the tumor is also impaired (Hamzah et al. 2008). Importantly, tumor response to therapy is also impacted. Hypoxia is known to reduce tumor cell sensitivity to radiation and chemotherapy (Teicher 1996), and the delivery of systemically administered cytotoxics into tumors is dramatically impeded, especially in areas of low blood flow and raised tumor IFP (Jain 1989; Wildiers et al. 2003; Tong et al. 2004). It is thus clear that tumor angiogenesis fuels a cascade of microenvironmental abnormalities which can hinder therapeutic outcomes. Reversal of these abnormalities, achieved through “normalizing” tumor vessels, is therefore an intuitively attractive proposition.

THE VASCULAR NORMALIZATION HYPOTHESIS

The vascular abnormalities in tumors stem from an imbalance between proangiogenic and antiangiogenic factors in the local milieu (Jain 2005b). The “vascular normalization” hypothesis states that direct or indirect antiangiogenic therapy (usually therapy aimed to neutralize VEGF activity) tips the imbalance between pro- and antiangiogenic factors back toward equilibrium (Fig. 1). As a result, vessel structure and function become more normal: vessels are more mature with enhanced PVC coverage, blood flow is more homogeneous, vessel permeability and hypoxia are reduced, and importantly the delivery of systemically administered anticancer therapies into tumors is more uniform. This concept explains why antiangiogenic therapy’s benefit has often been observed when it is given with chemotherapy (Table 1; Jain 2001, 2005b).

Figure 1.

Proposed role of vessel normalization in the response of tumors to antiangiogenic therapy. (A) Tumor vasculature is structurally and functionally abnormal. It is proposed that antiangiogenic therapies initially improve both the structure and the function of tumor vessels. However, sustained or aggressive antiangiogenic regimens may eventually prune away these vessels, resulting in a vasculature that is both resistant to further treatment and inadequate for the delivery of drugs or oxygen. (B) Dynamics of vascular normalization induced by VEGFR2 blockade. On the left is a two-photon image showing normal blood vessels in skeletal muscle; subsequent images show human colon carcinoma vasculature in mice at day 0 and day 3 after administration of VEGR2-specific antibody. (C) Diagram depicting the concomitant changes in pericyte (green) and basement membrane (blue) coverage during vascular normalization. (D) These phenotypic changes in the vasculature may reflect changes in the balance of pro- and antiangiogenic factors in the tissue. (Figure adapted from Jain 2005; reprinted with permission from the American Association for the Advancement of Science © 2005.)

Table 1.

Actual and potential therapeutic benefits of vascular normalization in solid tumors

Therapy	Benefits	Comments
Antiangiogenic monotherapy	Reduction in tumor-associated edemaa	Benefit maximized for intracranial tumors (primary tumors and brain metastases)
	Fewer metastases because of a reduction in primary tumor cell shedding into the circulationb	Showed in genetic models but not with pharmacologic normalizationc
	Fewer metastases because of reduced hypoxia and hence a reduction in EMTd	Showed in genetic models but not with pharmacologic normalizationc
	Improved infiltration of native antitumor immune cellse
Antiangiogenic therapy plus radiotherapy	Improved tumor controlf	Radiotherapy should be given during the normalization window when hypoxia is reduced
Antiangiogenic therapy plus systemic chemotherapy	Improved tumor control because of increased drug delivery into tumorsg	Showed preclinically but limited clinical evidence to date
Antiangiogenic therapy plus immunotherapy	Improved tumor control because of increased immune cell infiltration and normalization of the microenvironmenth	Showed preclinically but limited clinical evidence to date

Abbreviations: EMT, epithelial-to-mesenchymal transition.

^aKamoun et al. 2009.

^bXian et al. 2006; McCarty et al. 2007; Mazzone et al. 2009.

^cThis is controversial given recent reports of increased metastasis after antiangiogenic therapy in certain animal models (Ebos et al. 2009; Paez-Ribes et al. 2009).

^dMazzone et al. 2009.

^eGriffioen et al. 1996; Dirkx et al. 2006.

^fGorski et al. 1999; Hansen-Algenstaedt et al. 2000; Lee et al. 2000; Kozin et al. 2001; Ader et al. 2003; Winkler et al. 2004; Ansiaux et al. 2005, 2006; Pore et al. 2006a; Dings et al. 2007; Kashiwagi et al. 2008; Batra et al. 2009; Cerniglia et al. 2009; Tsukada et al. 2009; McGee et al. 2010.

^gTeicher et al. 1995a,b; Wildiers et al. 2003; Segers et al. 2006; Dickson et al. 2007a,b,c; Bhattacharya et al. 2008, 2009; Zhou et al. 2008; Juan et al. 2009; Zhou and Gallo 2009; J Liu, S Liao, B Diop-Frimpong, et al., unpubl. data.

^hHuang et al. 2002; Li et al. 2006; Manning et al. 2007; Hamzah et al. 2008; Shrimali et al. 2010.

EVIDENCE FOR VASCULAR NORMALIZATION IN TUMORS

Preclinical Evidence

Since we proposed the normalization concept, a large number of preclinical studies have confirmed the presence of the “normalized” vessel phenotype through restoring equilibrium between pro- and antiangiogenic signaling: either through suppression of angiogenic factors or enhancement of antiangiogenic activity (Table 2). As discussed below, many studies testing the effects of antiangiogenic drugs have shown a presence of a “normalization window”—a time period beginning with the appearance of a normalized vascular phenotype (typically within 1–2 days of starting treatment), and ending when features of normalization are lost (Tong et al. 2004; Winkler et al. 2004; Jain 2005b; Batchelor et al. 2007; Kamoun et al. 2009). The window’s closure may be caused by excessively high or prolonged dosing of antiangiogenics (tipping the balance in favor of antiangiogenic factors and hence leading to vascular regression), or because of the emergence of resistance to antiangiogenic therapy, in which tumors recruit vessels via alternate modes. The concept of normalization of tumor vessels has evolved dramatically over time. Interestingly, many genetic studies have not shown this window but rather show prolonged maintenance of vascular normalization (e.g., see Mazzone et al. 2009). Whether this can be realized using pharmacological agents is an open question. Nonetheless, these studies have provided exciting insights into the molecular underpinnings of vessel structure and function in solid tumors, and give hope for the strategic design of therapies giving rise to enduring normalization. In this section, we discuss the key molecules implicated in tumor vessel biology with a focus on their role in regulating vascular normalization.

Table 2.

The number of preclinical studies demonstrating features of vascular normalization in the published literature

	Direct antiangiogenics	Indirect antiangiogenics	Genetic models	Total
Structure
Reduced vessel diameter	10a	4b	5c	19
Increased pericyte coverage/proximity	14d	3e	13f	30
Normalized basement membrane	5g	0	1h	6
Function
Improved oxygenation	9i	12j	9k	30
Reduced permeability/IFP	12l	8m	5n	25
Improved delivery of drugs or systemically administered molecules	8o	5p	3q	16

^aYuan et al. 1996; Tong et al. 2004; Dickson et al. 2007b; Taguchi et al. 2008; Falcon et al. 2009; Juan et al. 2009; Kamoun et al. 2009; Chae et al. 2010; Koh et al. 2010; Primo et al. 2010.

^bJain et al. 1998; Izumi et al. 2002; Delmas et al. 2003; Qayum et al. 2009.

^cHamzah et al. 2008; di Tomaso et al. 2009; Nasarre et al. 2009; Van de Veire et al. 2010; Rolny et al. 2011.

^dInai et al. 2004; Tong et al. 2004; Nakahara et al. 2006; Dickson et al. 2007b; Dings et al. 2007; Fischer et al. 2007; Zhou et al. 2008; Falcon et al. 2009; Juan et al. 2009; Kamoun et al. 2009; Ohta et al. 2009; Zhou and Gallo 2009; Chae et al. 2010; Primo et al. 2010.

^eSalnikov et al. 2005; Bhattacharya et al. 2008; Qayum et al. 2009.

^fBenjamin et al. 1999; Dickson et al. 2007a,c; Greenberg et al. 2008; Hamzah et al. 2008; Kashiwagi et al. 2008; Stockmann et al. 2008; di Tomaso et al. 2009; Maione et al. 2009; Mazzone et al. 2009; Nasarre et al. 2009; Rolny et al. 2011; J Liu, S Liao, B Diop-Frimpong, et al., unpubl. data.

^gTong et al. 2004; Winkler et al. 2004; Zhou et al. 2008; Kamoun et al. 2009; Zhou and Gallo 2009.

^hMazzone et al. 2009.

ⁱHansen-Algenstaedt et al. 2000; Lee et al. 2000; Winkler et al. 2004; Dings et al. 2007; Fischer et al. 2007; Eichhorn et al. 2008; Batra et al. 2009; Skuli et al. 2009; McGee et al. 2010.

^jTeicher et al. 1995b; Jain et al. 1998; Bernsen et al. 1999; Cohen-Jonathan et al. 2001; Delmas et al. 2003; Ansiaux et al. 2005, 2006; Pore et al. 2006b; Segers et al. 2006; Cerniglia et al. 2009; Qayum et al. 2009; Cham et al. 2010.

^kAder et al. 2003; Hamzah et al. 2008; Kashiwagi et al. 2008; Stockmann et al. 2008; Maione et al. 2009; Mazzone et al. 2009; Skuli et al. 2009; Tsukada et al. 2009; Rolny et al. 2011.

^lYuan et al. 1996; Lee et al. 2000; Wildiers et al. 2003; Tong et al. 2004; Nakahara et al. 2006; Dickson et al. 2007b; Kurozumi et al. 2007; Taguchi et al. 2008; Zhou et al. 2008; Kamoun et al. 2009; Ohta et al. 2009; Zhou and Gallo 2009.

^mJain et al. 1998; Izumi et al. 2002; Ansiaux et al. 2005; Salnikov et al. 2005; Bhattacharya et al. 2008, 2009; Schnell et al. 2008; Cerniglia et al. 2009.

ⁿDickson et al. 2007c; Kashiwagi et al. 2008; di Tomaso et al. 2009; Mazzone et al. 2009; Rolny et al. 2011.

^oWildiers et al. 2003; Tong et al. 2004; Nakahara et al. 2006; Dickson et al. 2007b; Kurozumi et al. 2007; Zhou et al. 2008; Juan et al. 2009; Zhou and Gallo 2009.

^pTeicher et al. 1995a,b; Segers et al. 2006; Bhattacharya et al. 2008, 2009.

^qDickson et al. 2007a,c; J Liu, S Liao, B Diop-Frimpong, et al., unpubl. data.

VEGF

To date, all therapies approved as treatment for solid tumors target the proangiogenic action of VEGF. Preclinical studies have used a number of approaches including (1) specific VEGF blockade with agents that interfere with VEGF binding to its receptors (including antihuman VEGF antibodies such as A4.6.1 and bevacizumab), antimurine VEGF antibodies, and the “VEGF-Trap” aflibercept; (2) inhibition of VEGFR2 function (using anti-VEGFR2 antibodies such as DC101 or receptor tyrosine kinase inhibitors [TKIs] which inhibit the kinase domain of VEGFRs). In addition, elegant preclinical work examining the role of VEGF has come from genetic studies, which provide a cleaner understanding of the effect of a single factor in specific cell populations, and include inducible and/or conditional models.

Our laboratory provided the first evidence of vascular normalization in response to VEGF blockade in 1996 (Yuan et al. 1996). We examined the effects of A4.6.1 therapy on various different human tumor types implanted in mice under surgically created transparent windows, allowing serial microscopic and dynamic imaging of the tumor vessels’ structure and function in real time. Vascular diameter and tortuosity were both reduced after a single dose of A4.6.1, accompanied by a drop in permeability. These changes were dynamic and reversible, and permeability rose to baseline again 5 days after treatment (Yuan et al. 1996). If therapy was continued, however, features of normalization were soon replaced by marked vascular regression, likely because of excessive neutralization of VEGF activity. These findings provided early evidence that neutralization of tumor-cell-derived VEGF could reverse, albeit transiently, some of the abnormalities of the tumor microvasculature.

Since this report, a vast array of studies has explored changes in vessel structure and function in response to VEGF blockade in greater detail. Structural changes such as reduced vessel density have been consistently observed with anti-VEGF antibody therapy and aflibercept in a wide variety of tumor models (Hansen-Algenstaedt et al. 2000; Lee et al. 2000; Wildiers et al. 2003; Inai et al. 2004; Tong et al. 2004; Dickson et al. 2007b; Juan et al. 2009). This has generally been accompanied by an increase in vascular maturity (the proportion of PVC-covered vessels), such that the treated vasculature resembles that of normal tissue more closely (Yuan et al. 1996; Inai et al. 2004; Tong et al. 2004; Dickson et al. 2007b). In some reports, another abnormal feature of tumor vessels—the haphazard and disorganized basement membrane—was also normalized after anti-VEGF therapy (Tong et al. 2004; Winkler et al. 2004; Kamoun et al. 2009).

These structural changes are often accompanied by normalization of vessel function. Antibody therapy against the VEGF-VEGFR pathway has been shown to reduce vessel permeability, lowering intratumoral IFP, and improving perfusion (Table 2). This can reduce tumor hypoxia improving tumor radiosensitivity (Gorski et al. 1999; Lee et al. 2000; Winkler et al. 2004; Dings et al. 2007), improve penetration of large molecules into the tumor interstitium (Tong et al. 2004), and heighten delivery of systemically administered chemotherapeutics into tumors (Wildiers et al. 2003; Dickson et al. 2007b). Of interest, anti-VEGF antibody therapy in melanoma models has also been shown to improve the function of tumor immunotherapy, by facilitating increased delivery of antitumor cytotoxic T cells into the tumor parenchyma (Shrimali et al. 2010).

Such studies have highlighted the presence of the normalization window, and emphasized its therapeutic importance. In many cases, normalization after VEGF blockade is first noted within hours to days of therapy commencement, and is short-lived (lasting for 7–10 days) (Winkler et al. 2004). It is during this window period that enhanced radiotherapy sensitivity (and hence tumor control) is attained (Winkler et al. 2004). Other groups have shown similar principles with chemotherapy, showing improved tumor control with combined cytotoxics and anti-VEGF agents in a schedule-dependent manner (i.e., chemotherapy delivery and tumor control are optimized if chemotherapy is given during the normalization window) (Table 1).

As opposed to antibodies, VEGFR TKI’s work at an intracellular level and all have some nonspecific “off-target” binding to other members of the kinome including those present in tumor cells. As a result, mechanistic interpretation of their effects on tumors is more difficult. Although almost all initial clinical trials combining chemotherapy with TKIs yielded disappointing results, recent success from this approach has been reported in a randomized phase 3 trial using an anti-VEGFR TKI with low inhibitory potency (Herbst et al. 2010). Moreover, structural aspects of normalization has been observed in preclinical studies after VEGFR TKI therapy including reduction in vessel density, increased vessel maturity, and a tighter proximity of PVCs to ECs (Table 2). In addition, basement membrane abnormalities can also be partially restored in models of GBM treated the anti-VEGFR TKI cediranib (Kamoun et al. 2009). Similarly, functional alterations including reduced vessel permeability and IFP, increased blood flow, improved delivery of systemically administered antibodies and chemotherapy, and increased oxygenation and radiosensitivity have been reported (Table 2). In studies of GBM treated with the TKI sunitinib, chemotherapy delivery into tumors was correlated directly with the extent to which features of vascular normalization were attained (Zhou and Gallo 2009).

One important finding of translational relevance is that by reducing vessel leakiness, anti-VEGF therapy can reduce tumor-associated edema. This is particularly important for brain tumors, in which the tight confines of the intracranial space provide marked spatial limitations. Indeed, the anti-VEGFR TKI cediranib, when given to mice with GBM is able to prolong survival dramatically purely though this mechanism. Although no reduction in tumor growth is observed, the reduction in edema delays a precipitous increase in intracranial pressure (Kamoun et al. 2009).

Studies employing genetic models shed further light onto mechanisms behind vessel normalization. When VEGF^−/− tumors are grown in wild-type animals, tumor vessels show improved PVC coverage (Greenberg et al. 2008). Similarly, when wild-type tumors are grown in mice with a VEGF^−/− myeloid cell population, tumors again show greater vessel maturity, reduced hypoxia, and chemosensitivity (Stockmann et al. 2008). It thus appears that VEGF-induced vascular normalization is a generalized phenotype, regardless of the source of VEGF.

Several important lessons arise from the above reports, which also give rise to further unanswered questions. First is the potential importance of judicious dosing of anti-VEGF therapy (i.e., doses high enough to reverse vessel abnormalities but not so high as to cause excessive vessel regression). Given that normalization depends on achieving a balance between pro and antiangiogenic factors, this concept is somewhat intuitive but has been emphasized particularly in studies of sunitinib treatment of GBM in which only low (but not high) doses of anti-VEGF treatment improved chemotherapy delivery into tumors (Zhou et al. 2008; Zhou and Gallo 2009). Second, certain molecular mechanisms of anti-VEGF therapy-mediated normalization have been uncovered. For example, in the case of GBM, it appears that tumor cell-derived angiopoietin-1 is an absolute requirement for normalization, and that therapy-induced activation of matrix metalloproteinases (MMPs) drives basement membrane degradation leading to the normalization of basement membrane structure (Winkler et al. 2004). Third, the precise effect of anti-VEGF related normalization on tumor growth is still controversial. In many studies, therapy induces a tumor growth delay, but this may well relate to vascular pruning and regression rather than normalization (Kim et al. 1993). Others have shown an increase in the rate of tumor growth accompanying normalization (Stockmann et al. 2008). This could be caused by the improved availability of oxygen and nutrients, but the vast majority of studies revealing improved tumor perfusion and oxygenation after VEGF inhibition have not replicated this finding. Differences could relate to variations in oxygen and glucose consumption rates of different tumors (Gullino 1982).

The Angiopoietin-Tie2 Axis

The angiopoietin-Tie2 pathway is perhaps the second most important EC-specific pathway regulating both healthy and tumor-associated angiogenesis (Suri et al. 1996; Huang et al. 2010). Angiopoietin-1 (Ang-1, produced primarily by PVCs), and Ang-2, produced primarily by ECs, serve as ligands for the EC Tie-2 receptor. Although their precise actions are complex and context-dependent, Ang-1 is generally considered a Tie-2 agonist which promotes vessel maturity and stability and reduces leakiness (Thurston et al. 1999, 2000; Augustin et al. 2009) and Ang-2 serves as a competitive antagonist for Tie-2, enhancing tumor angiogenesis and destabilizing blood vessels (Chae et al. 2010).

In keeping with these actions, pharmacological blockade of Ang-2 (but not Ang-1) is able to normalize the tumor vessel phenotype, improving vascular PVC coverage and tightening EC junctions (Falcon et al. 2009). Similarly, wild-type tumors grown in Ang2^−/− mice show an increase in PVC coverage, a reduction in vessel diameter, and a delayed early phase of tumor growth (Nasarre et al. 2009). Ang-2 inhibition operates not only through preventing Ang-2/Tie-2 interaction on ECs, but might also interfere with the proangiogenic activities of Tie-2 expressing monocytes (TEMs) (Coffelt et al. 2010; Mazzieri et al. 2011).

Recently, a dual pharmacological inhibitor of VEGF and the angiopoietins (named the “double antiangiogenic protein,” DAAP) (Koh et al. 2010) was shown to inhibit the growth of several murine tumors and, in the case of implanted ovarian carcinoma, resulted in a normalization of vessels. This is likely to be the first of many studies examining the strategy of inhibiting multiple proangiogenic factors to attain a greater degree of vessel normalization.

Placental Growth Factor

Placental growth factor (PlGF) is another member of the VEGF family that has recently been implicated in tumor angiogenesis and as a regulator of the abnormal tumor vessel phenotype. PlGF is not significantly expressed by normal tissues, but is found abundantly in various tumors (Fischer et al. 2007), and plays an important role in tumors through stimulation of EC growth, mobilization of bone marrow cells, and recruitment of macrophages (Carmeliet et al. 2001) through its binding to VEGFR1 and its coreceptors NRP1 and 2.

Pharmacological PlGF blockade has yielded conflicting results, with some reports demonstrating a profound effect on tumor growth accompanied by angiogenesis inhibition, vessel maturation, resolution of hypoxia, and improved efficacy of chemotherapy (Fischer et al. 2007; Van de Veire et al. 2010), and others demonstrating limited effects on tumor growth (Bais et al. 2010). Recent reports using PlGF^−/− mice and si-RNA silencing of PlGF in established tumors suggest that PlGF inhibition can indeed improve vessel structure and induce a tumor growth delay (Van de Veire et al. 2010). The histidine-rich glycoprotein (HRG), through its down-regulation of PlGF, may mediate this process, inducing vessel normalization and also modifications in tumor-associated macrophages from a protumor (M2) to antitumor (M1) phenotype (Huang et al. 2011; Rolny et al. 2011).

Oxygen Sensors

Given the critical interplay between tumor hypoxia and angiogenesis, modulation of tumor-oxygen sensing has also proven an effective strategy to normalize tumor vessels. First, and not surprisingly, down-regulating tumor HIF-1α expression inhibits tumor angiogenesis and might improve the efficacy of chemotherapy (Liu et al. 2008). In addition, the EC oxygen sensor PHD2 also plays an important role in generating the abnormal tumor vessel phenotype (Mazzone et al. 2009). Cells in PHD2^+/− mice, by expressing PHD2 at half normal levels, behave as if they sense lower oxygen tensions and as such are preadapted to hypoxia. Syngeneic tumors grown in these mice have normalized vessels, with tight EC junctions (because of a HIF-2α-induced increase in VE-Cadherin expression), a mature PVC phenotype, reduced hypoxia, and less leakiness. These effects are also seen in mice with an EC-specific single allele deletion of PHD2, suggesting that hypoxia (and hence PHD2 inhibition) drives ECs toward a normalized phenotype (as opposed to the effects of hypoxia on tumor cells which promotes VEGF expression and an abnormal vessel phenotype) (Mazzone et al. 2009).

The PHD2 studies also show another important consequence of normalized vessels: Tightened EC junctions in PHD2^−/− mice lead to a reduction in shedding of primary tumor cells into the circulation, with a consequent decrease in the numbers of circulating tumor cells and metastasis formation (in the absence of any effect on primary tumor growth) (Mazzone et al. 2009). Although exciting and of potential clinical relevance, it should be noted that no pharmacologic intervention geared toward normalizing vessels has been able to achieve this goal.

Perivascular Cell Targets

A number of molecules regulate PVC recruitment to vessels including Ang-1, PDGFRβ, transforming growth factor (TGF)-β, and sphingosine-1-phosphate (Jain 2003). Already discussed in this article are the importance of Ang-1 and PDGFRβ signaling in maintaining normalized vessels. More recently, the regulator of G-protein signaling 5 (Rgs5), a molecule involved in PVC differentiation and a specific marker of PVCs, has been implicated as a key mediator of vascular normalization (Hamzah et al. 2008). Although the function of Rgs5 is poorly understood, tumors growing in Rgs5^−/− mice have a normalized vasculature, showing reduced vessel diameter and structural irregularity, improved oxygenation, and less vessel permeability. Moreover, adoptive transfer of activated immune cells into tumors of Rgs5^−/− mice yields increased antitumor T lymphocyte trafficking and a significant improvement in mouse survival (Hamzah et al. 2008).

Other Angiogenic Molecules

A number of other critical molecules involved directly in tumor angiogenesis are also contributors to the abnormal vessel phenotype in solid tumors. These include the EC-specific integrins (Friedlander et al. 1995; Kurozumi et al. 2007; Skuli et al. 2009; Desgrosellier and Cheresh 2010; Primo et al. 2010), semaphorins (Bielenberg et al. 2006; Maione et al. 2009), members of the nitric oxide synthase family (Kashiwagi et al. 2005, 2008; Yu et al. 2005), TGF-β (Liu et al., submitted for publication), and interferon-β (Dickson et al. 2007a,c; Taylor et al. 2008). In keeping with the notion that it is the balance between pro- and antiangiogenic stimuli that moderates the equilibrium between normal and abnormal vessels, modulation of the activity of these molecules is able to normalize tumor vessels structurally and/or functionally in a variety of tumor models preclinically.

Indirect Inhibitors of Angiogenesis

Unlike the strategies described above, which have direct effects on tumor angiogenesis, a number of drugs affect tumor vessels indirectly. These “indirect” antiangiogenics often inhibit pathways that drive tumor cell proliferation which are coincidentally upstream of pro- or antiangiogenic molecules (Vogelstein and Kinzler 2004). Examples include inhibitors of tumor cell oncogenes, endocrine therapies for sex hormone-dependent tumors, and metronomic chemotherapy.

It is well known that oncogenic activation not only drives tumor cell division but often has multiple other downstream effects including promotion of sustained angiogenesis (Hanahan and Weinberg 2000), either through up-regulation of factors such as VEGF or down-regulation of antiangiogenic molecules such as thrombospondin. As a consequence, inhibitors of oncogene activity can indirectly normalize tumor vessels, potentially enhancing not only the action of chemotherapy and radiotherapy, but also their own antitumor cell activity. Investigators have reported various aspects of normalization in response to inhibition of several oncogenes including HER-2 (Izumi et al. 2002), the PI3K-AKT-mTOR axis (Pore et al. 2006a; Schnell et al. 2008; Xue et al. 2008; Qayum et al. 2009), Ras (Cohen-Jonathan et al. 2001; Delmas et al. 2003; Qayum et al. 2009), and the epidermal growth factor receptor (EGFR) (Cerniglia et al. 2009; Qayum et al. 2009; Johns et al. 2010). In the case of HER-2, we have shown that treatment of leptomeningeal HER-2 positive breast cancer with trastuzumab (a monoclonal antibody directed against HER-2) not only inhibits tumor cell proliferation, but also normalizes vessels. These changes occur because HER-2 inhibition modifies the expression of five angiogenesis-related molecules, tipping the pro- versus antiangiogenic balance toward equilibrium (Izumi et al. 2002).

Results of EGFR inhibition (initially designed to target oncogenic EGFR mutations in diseases such as nonsmall cell lung cancer) have also provided interesting results. EGFR inhibitors such as gefitinib and erlotinib normalize tumor vessel structure and function in several mouse models of solid tumors (Cerniglia et al. 2009; Qayum et al. 2009) and synergize with chemotherapy and/or radiotherapy to maximize tumor control. Importantly, these studies (and others pertaining to oncogene inhibitors) report normalization of longer duration than that observed with anti-VEGF therapy (Qayum et al. 2009), potentially relating to specific suppression of oncogene-driven, excessive VEGF within a tumor rather than total VEGF suppression from anti-VEGF therapy, which ultimately causes vascular regression.

Depriving hormone-dependent tumors of the sex hormones on which they depend represents another mode of indirect antiangiogenic therapy that can normalize vessels. We have shown that castration of mice bearing the androgen-dependent Shionogi carcinoma indirectly reduces tumoral VEGF expression, in turn inducing EC apoptosis, normalizing vessels structurally and functionally, and improving tumor oxygenation (Jain et al. 1998; Hansen-Algenstaedt et al. 2000).

A third important method of indirect normalization is “metronomic chemotherapy,” a treatment schedule where traditional cytotoxic drugs are given in low doses on a continuous schedule, the goal being to kill tumor ECs (Browder et al. 2000; Kerbel and Kamen 2004) and hence yield an antiangiogenic effect. It seems that at least part of the benefit from metronomic therapy may relate to vessel normalization. ECs exposed to low dose chemotherapy in vitro up-regulate expression of the antiangiogenic thrombospondin-1 (Bocci et al. 2003). As such, metronomic therapy may help to restore the equilibrium between pro- and antiangiogenic factors within tumors. As further support for this notion, metronomic gemcitabine has been shown to reduce vessel density and improve tumor oxygenation in pancreatic cancer xenografts, when compared to gemcitabine given on a conventional schedule (Cham et al. 2010). This normalization correlated with reductions in the levels of several proangiogenic factors.

Clinical Evidence for Vascular Normalization

Given the growing body of preclinical evidence suggesting vascular normalization as a potential mechanism of benefit from antiangiogenic therapy, we proceeded to investigate whether similar phenomena occur in human patients (Table 3). Such translational studies are fraught with complexity, requiring adequate numbers of patients who must undergo invasive and noninvasive investigations (often including serial tumor biopsies). Despite these challenges, a number of completed studies have now provided evidence for features of vascular normalization in clinical subjects, in particular disease settings.

Table 3.

Clinical studies demonstrating evidence of vascular normalization in human tumors

Tumor type	Antiangiogenic therapy	Changes in vessel structure	Changes in vessel function	Clinical observations
Primary tumors
Rectal carcinoma (n = 32)	Bevacizumab	↓Vessel density, ↑PVC coverage	↓Tumor blood flow, ↓IFP, improved delivery of FDG per vessel	Tumors became pale (Willett et al. 2004, 2009)
Glioblastoma (n = 31)	Cediranib	↓Vessel size	↓Permeability	↓tumor-associated edema, reduced patient need for corticosteroids (Batchelor et al. 2007, 2010)
High grade glioma (n = 5)	Bevacizumab	↓Vascular arcades and glomeruloid vessels		Fischer et al. 2008
Prostate carcinoma (n = 10)	Androgen ablation	Pruning of immature vessels, ↑PVC coverage		Benjamin et al. 1999
Metastatic disease
HER2+ breast cancer brain metastases (n = 22)	Lapatinib (indirect antiangiogenic)	↓Vessel tortuosity		Bullitt et al. 2007

The first study was a Phase 1/2 trial in a series of 32 patients with locally advanced rectal carcinoma (Fig. 2) (Willett et al. 2004, 2005, 2009). A single dose of bevacizumab monotherapy normalized tumor vessels within 12 days: (1) after bevacizumab, tumors appeared more pale and less hyperemic, accompanied by a 40% reduction in tumor blood flow on CT scan and a histological drop in vessel density; (2) tumor IFP fell by >50%; (3) vessel PVC coverage increased. Importantly, despite the reduction in vessel density and blood flow, uptake of the radionuclide 18-fluordeoxyglucose on PET scanning did not fall, suggesting that remaining vessels had improved functionality (Willett et al. 2004). More crucially, the pretreatment level of sVEGFR1, a marker of vascular normalization, correlated with treatment outcome and toxicity (Duda et al. 2010).

Figure 2.

Direct effects of antiangiogenic therapy in human patients with locally advanced adenocarcinoma of the rectum. (A) Six patients were treated with locally advanced rectal cancer underwent sigmoidoscopy before (upper panels) and 12 days after (lower panels) a single dose of bevacizumab (antihuman VEGF antibody). Tumors appear notably less hyperaemic after treatment, associated with a quantifiable decrease in tumor blood flow. (B) Positron emission tomography (PET) scanning using fluoro-deoxyglucose (FDG). Despite the reduction in tumor blood flow, the amount of extravasated FDG is similar before (left panel) and after (right panel) bevacizumab treatment, implying improved functionality of surviving vessels. (C) Graphical representation of FDG uptake on PET scanning for six patients. Again, there is no difference in tumor uptake of FDG between pretreatment values and those 12 days after a single dose of bevacizumab. (Figure adapted from Willett et al. 2004; reprinted, with permission, from Nature Publishing Group © 2004.)

A second study examined the effects of cediranib in 31 patients with recurrent GBM (Batchelor et al. 2007, 2010; Sorensen et al. 2009). Because serial tissue biopsies cannot be obtained from recurrent GBM patients, advanced “vascular” MRI was used to assess changes in vessel structure and function. Consistent with preclinical models, cediranib induced a rapid reduction in tumor vessel size vessel permeability. This reduction in permeability led to an attenuation of tumor-associated edema and hence the need for corticosteroid therapy. We developed a mathematical index of vascular normalization (comprising various indirect markers of normalization) and found this to be a strong predictor of individual patient outcomes (Sorensen et al. 2009). In addition to these findings, studies reporting results of serial GBM biopsy and autopsy specimens before and after anti-VEGF therapy have suggested a pattern of structurally more normal vessels after therapy (Fischer et al. 2008; di Tomaso et al. 2011).

VASCULAR ABNORMALITIES AND THERAPEUTIC NORMALIZATION IN NONMALIGNANT DISEASES

The normalization hypothesis was first supported in the context of the solid tumor vasculature. Recently, however, an explosion in angiogenesis research has revealed that similar vascular abnormalities are seen in a variety of nonmalignant diseases. These observations, coupled with a heightened understanding of the deleterious effects of an abnormal vasculature, have led to a number of studies exploring the benefits of antiangiogenic therapy-induced vascular normalization in a variety of common and rare benign diseases.

Benign Tumors

Although lacking metastatic potential, benign tumors still depend on angiogenesis to fuel their persistent growth, and the balance between pro- and antiangiogenic factors plays an important role in their progression. A well-characterized example is the vestibular schwannoma (acoustic neuroma), a benign hyperproliferation of the Schwann cells that line peripheral nerves. Neurofibromatosis 2, a rare genetic disease defined by a defect in a single allele of the Nf2 gene (and hence loss of its product, the tumor suppressor merlin) often presents with bilateral acoustic neuromata, which compress the vestibular nerve causing deafness and other complications relating to their expansion in the cerebellopontine angle (Lu-Emerson and Plotkin 2009).

Vestibular schwannomas are known to express VEGF and VEGFRs, and the VEGF level within these tumors correlates with their growth rate (Brieger et al. 2003; Caye-Thomasen et al. 2003, 2005; Plotkin et al. 2009). Interestingly, the mechanism of heightened VEGF activity in these tumors is not hypoxia-driven VEGF overexpression, but rather a loss of factors that attenuate VEGF function. In detail, merlin deficiency in schwannoma cells is associated with a concomitant down-regulation of SEMA 3D, SEMA 3F, and SEMA 3G (Wong et al. 2010). These semaphorins normally act via neuropilin receptors as negative regulators of VEGF activity, and loss of their antiangiogenic influence drives robust angiogenesis in schwannomas.

In preclinical studies, we have treated orthotopic schwannomas derived from both human schwannoma cells and murine NF2 null cells with anti-VEGF agents (Wong et al. 2010). Therapy reproduced many aspects of vascular normalization, including a prompt reduction in tumor vessel permeability, and a reduction in vessel surface area seen by day 6 of treatment. As anti-VEGF therapy tipped the balance between pro- and antiangiogenic factors toward normal, structural features of normalization including an increase in PVC coverage were also observed. In keeping with the known correlation between VEGF activity and growth rate in these tumors, anti-VEGF therapy inhibited tumor growth, an effect mediated primarily through an antiangiogenic effect (Wong et al. 2010). As proof of concept, we have also shown that reintroduction of SEMA3F into NF2 null cells reduces the growth of implanted tumors, which show a reduction in vessel density and improved PVC coverage (HK Wong, A Shimizu, ND Kirkpatrick, et al., unpubl. data). Mice-bearing NF2^−/−(SEMA3F) tumors in the brain also have prolonged survival compared with those bearing NF2^−/− tumors without SEMA3F.

These features have been emphasized through the results of a clinical trial in NF2 patients (Plotkin et al. 2009). In this study, semaphorin down-regulation was observed in patient acoustic neuroma specimens, and treatment with bevacizumab resulted in a 60% reduction in tumor growth, and a corresponding improvement in hearing acuity. Importantly, the intratumoral apparent diffusion coefficient on MRI (ADC, a measure of the magnitude of water diffusion) correlated with the degree of tumor shrinkage, suggesting that patients with excess edema are most likely to benefit from bevacizumab treatment. These clinical findings are supported by the preclinical data, and confirm the benefit of vessel normalization in this benign tumor. Delaying tumor growth in this disease is likely to delay the need for more aggressive therapies such as surgery to prevent tumor-associated morbidity.

Age-Related Macular Degeneration

Perhaps the most successful application of antiangiogenic therapy has been in the treatment of age-related macular degeneration (ARMD), the leading cause of blindness in the United States (Bressler 2009a,b). “Wet” ARMD, the less common but more disabling form of the disease, has as its hallmark excessive choroidal angiogenesis, seen beneath the retinal pigment epithelium (RPE) and also between the RPE and overlying retina. Several proangiogenic factors serve as players in the retinal microenvironment (including FGF, PDGF, and PlGF), but VEGF overexpression by RPE cells (caused by focal hypoxia and abnormal thickening of Bruch’s membrane) is the most important (Bressler 2009b; Rajappa et al. 2010). VEGF abundance results in a retinal vasculature resembling that of solid tumors, characterized by focal macular edema and retinal hemorrhage.

Traditional therapy for wet ARMD involves laser photocoagulation of proliferative vessels, and pharmacologic therapy has proved an elusive goal until recently. Given the crucial role for VEGF in this disease, two anti-VEGF therapeutics have been developed as treatment of wet ARMD with great success leading to their approval for clinical use by the U.S. FDA. The two main agents are ranibizumab (a humanized monoclonal antibody fragment derived from the same murine antibody as bevacizumab) and pegaptanib (a pegylated aptamer that binds to VEGF). Both are administered by serial intravitreal injections. In the case of pegaptanib, preclinical work shows features of vascular normalization in response to treatment including a reduction in microvascular leakage, leukocyte adhesion, and choroidal neovascularization (Eyetech Study Group 2002; Ishida et al. 2003). Clinical observations also confirm a reduction in vascular leakage (as detected by fluorescein angiography) after treatment with anti-VEGF agents (Bressler 2009a). In turn, this normalized vessel phenotype dramatically alters the retinal microenvironment and has led to impressive clinical results (Gragoudas et al. 2004; Rosenfeld et al. 2006; Ferrara 2010), including improved visual acuity lasting for up to 24 mo after commencement of therapy.

Cutaneous Psoriasis

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of keratinocytes and angiogenesis, and is another nonmalignant disease in which abnormal angiogenesis is critical to disease progression. Current models suggest that angiogenesis is required for the induction of psoriatic pathology and maintains the immune and inflammatory processes that drive psoriasis (Heidenreich et al. 2009).

Cutaneous psoriasis starts with angiogenesis in the superficial dermal microvasculature. Dermal papillary capillaries increase in tortuosity, diameter and permeability, and show prominent elongation. Psoriatic plaques show characteristic features of venous capillaries, as opposed to normal skin in which capillary loops show an arterial phenotype (Braverman and Yen 1977). Besides these morphological changes, papillary dermal microvessels in psoriatic lesions show increased expression of inflammation-associated adhesion molecules such as E-selectin, Intercellular adhesion molecule-1 (ICAM-1) and vascular cell-adhesion molecule-1 (VCAM-1) (Das et al. 1994). These molecules allow adhesion of leukocytes to the endothelium and the establishment of an inflammatory response. Endothelial cells of psoriasis plaques also show enhanced proliferation (Creamer et al. 1997).

Underlying psoriatic angiogenesis, several proangiogenic factors including tumor necrosis factor-α (TNF-a), VEGF, HIFs, Interleukin (IL)-8, and angiopoietins, are all enriched in psoriatic skin (Heidenreich et al. 2009). As abnormal angiogenesis is considered to be the key to the initiation and progression of psoriasis, several antiangiogenic therapies have been developed to treat psoriasis. These agents either interfere either directly or indirectly with pathways regulating endothelial cell physiology, or directly targeting angiogenesis. Through attenuating proangiogenic stimuli, these agents act to reduce vascular abnormalities seen in psoriatic plaques and hence moderate disease progression.

The effects of VEGF blockade have been reported using genetic murine models of psoriasis. After therapy, a reduction in vessel density and diameter was observed, corresponding to a reduction in severity of skin lesions (Schonthaler et al. 2009). Clinical case reports have also suggested improvement in skin lesions after treatment with anti-VEGFR TKIs (Keshtgarpour and Dudek 2007; Fournier and Tisman 2010; Narayanan et al. 2010). More recently, nonviral somatic delivery of the recombinant disintegrin domain (RDD) of metargidin, which interferes with the VEGF pathway and angiogenesis, was shown to lower cutaneous blood flow in psoriasis-affected mice and reduce disease severity (Zibert et al. 2010). Given these data, prospective and controlled clinical trials are needed to evaluate the efficacy of these and other antiangiogenic therapies to determine the extent to which vessel normalization underpins the observed clinical benefit.

Hereditary Hemorrhagic Telangiectasia

Hereditary hemorrhagic telangiectasia (HHT), also known as Osler–Weber–Rendu syndrome, is a rare autosomal dominant genetic disease characterized by excessive and abnormal blood vessel formation. Patients with HHT harbor large numbers of vascular telangiectasias, most prominent on mucous membranes and skin, and as arteriovenous malformations (AVMs) in the brain, lung, and liver. These malformations comprise dilated, thin-walled networks of dysplastic capillaries that are inherently fragile and prone to bleeding, giving rise to symptoms such as epistaxis, excessive bruising, and potentially life-threatening visceral and intracranial hemorrhage.

Small case series have suggested a beneficial role for antiangiogenic therapy in HHT. Bevacizumab, for example, has been shown to dramatically reduce the frequency of epistaxis and blood transfusion requirements (Flieger et al. 2006; Bose et al. 2009). More recently, a translational study shed light on the mechanism of action of antiangigogenic therapy in HHT. Using a murine genetic model of HHT, Lebrin and colleagues found that the antiangiogenic agent thalidomide increased vessel maturity in telangiectatic regions (as evidenced by increased PVC coverage), hence normalizing vessels and attenuating vessel wall defects (Lebrin et al. 2010). This benefit is at least in part mediated by PDGF-B mediated recruitment of PVCs to ECs. Moreover, the same group found that treating HHT patients with thalidomide normalized vessels in the nasal mucosa and reduced epistaxis significantly.

Atherosclerotic Plaques

Alongside cancer, atherosclerosis represents a major cause of mortality worldwide. Although the role for angiogenesis in the progression of atherosclerotic plaque development is a young and relatively unexplored area, evidence does suggest that new vessel formation in the coronary plaque vasa vasorum is an important contributor to disease progression and plaque complications (Jeziorska and Woolley 1999; Kolodgie et al. 2003; Virmani et al. 2005; Jain et al. 2007). Indeed, hypoxia within plaques drives angiogenesis and an abnormal vasculature. The plaque microenvironment also contains high levels of eNOS and TNF-α. As a result, intra-plaque hemorrhage can occur (particularly in noncalcified plaques, which have the highest vessel density and are more prone to rupture), accelerating plaque progression and rupture, and leading to myocardial infarction (Gossl et al. 2010).

Preclinical studies in genetic models of atherosclerosis have examined the effects of angiogenesis inhibition on plaque biology. For example, the angiogenesis inhibitors endostatin and TNP-470 have been shown to not only reduce new vessel formation within plaques, but also slow plaque growth (Moulton et al. 1999). Although it might be possible that judicious use of antiangiogenic therapy could be used to normalize plaque vessels, potentially minimizing the risk of plaque hemorrhage and the attendant consequences, research is currently limited by the lack of suitable animal models to test appropriate agents, doses, and schedules.

NORMALIZATION OF THE LYMPHATIC VASCULATURE: AN EMERGING CONCEPT IN VASCULAR NORMALIZATION

Although normalization of tumor vessels is a concept largely pertaining to blood vessels, recent evidence suggests marked abnormalities in the function of solid tumor lymphatics. From the very early stages of tumor development, the lymphatic microvasculature becomes compressed and dysfunctional (Hagendoorn et al. 2006). The impaired function relates partly to compression, but also to valvular damage, impaired contractile activity, and occlusion by tumor cells (Padera et al. 2004). A good example is carcinoma of the ovary. Both mice and patients with disseminated ovarian cancer develop gross abdominal ascites, caused in part by impaired drainage of peritoneal fluid by regional lymphatics. We have recently found that diaphragmatic lymphatics in ovarian cancer–bearing mice have structural abnormalities not dissimilar from those found in tumor blood vessels, namely increased vessel density, diameter, and tortuosity (Liao et al. 2011). They also show impaired function with loss of valves and sluggish clearance of peritoneal fluid. Interestingly, when we inhibited the function of TGF-β, either pharmacologically or genetically, we observed normalization of lymphatic structure and function (Liao et al. 2011). TGF-β has known effects on both tumor cell biology and lymphangiogenesis, and the improved lymphatic function in such models may relate partly to relief of compression through a reduction in tumor burden. Such findings open avenues for future research into normalization of the lymphatic phenotype in tumors and other diseases characterized by lymphatic dysfunction.

SUMMARY

Research over several decades has clearly shown marked abnormalities in the tumor microvasculature, which have profound consequences for tumor growth, metastasis, and response to therapy. Although initial preclinical studies showed that anti-VEGF therapy is able to kill tumor vessels and destroy tumor vessels, the results of bevacizumab monotherapy in patients with solid tumors were overwhelmingly disappointing. Despite this, anti-VEGF treatments have been able to improve the efficacy of systemic chemotherapy, suggesting that they augment chemotherapy benefit in some way. The vascular normalization hypothesis helps to explain these findings. By restoring the balance between pro- and antiangiogenic factors in the tumor microenvironment, antiangiogenic therapy is able to restore a more normal tumor vasculature, capable of delivering cytotoxics more efficiently into tumors and enhancing the effects of radiotherapy. In recent years, a plethora of preclinical studies have shed light on the molecular regulators of this phenotypic switch.

Further understanding of the normalization process is required before it can be most effectively exploited in the clinical setting, which will be difficult given the great heterogeneity in tumor vessels within a single patient and between patients. First, as discussed above, the impact of vascular normalization on primary tumor growth requires further study. Second, further work is needed to resolve the controversy over whether antiangiogenic therapy through normalization serves to decrease the rate of solid tumor metastasis or through excessive vascular pruning serves to increase metastasis. Third, mechanisms of resistance to the normalization process should be explored, with the ultimate goal of developing pharmacologic therapies that can induce sustained vascular normalization in tumors. Fourth, more attention must be given to the wide range of benign diseases characterized by abnormal angiogenesis—a largely unexplored area potentially offering improved outcomes for millions of patients worldwide. Finally, biomarkers for vascular normalization are sorely needed to use this concept optimally in the clinical setting (Jain et al. 2009).

CONFLICTS OF INTEREST

R.K.J. has served in a consultant or advisory role to Astellas, AstraZeneca, Dyax, Genzyme, Noxxon Pharma, Regeneron, and SynDevRx, has equity in SynDevRx, has received honoraria from Genzyme, and has received research funding from AstraZeneca, Dyax, MedImmune, and Roche.