, Volume 10, Issue 3, pp 149–166

Angiogenesis and chronic inflammation: cause or consequence?


  • Carla Costa
    • Laboratory for Molecular Cell BiologyFaculty of Medicine of the University of Porto
    • Institute for Molecular and Cell Biology (IBMC)
    • iSEX, Association for the advanced study of human sexuality
  • João Incio
    • Department of BiochemistryFaculty of Medicine of the University of Porto
    • Department of BiochemistryFaculty of Medicine of the University of Porto
Review Paper

DOI: 10.1007/s10456-007-9074-0

Cite this article as:
Costa, C., Incio, J. & Soares, R. Angiogenesis (2007) 10: 149. doi:10.1007/s10456-007-9074-0


Evidence has been gathered regarding the association between angiogenesis and inflammation in pathological situations. These two phenomena have long been coupled together in many chronic inflammatory disorders with distinct etiopathogenic origin, including psoriasis, rheumatoid arthritis, Crohn’s disease, diabetes, and cancer. Lately, this concept has further been substantiated by the finding that several previously established non-inflammatory disorders, such as osteoarthritis and obesity, display both inflammation and angiogenesis in an exacerbated manner. In addition, the interplay between inflammatory cells, endothelial cells and fibroblasts in chronic inflammation sites, together with the fact that inflammation and angiogenesis can actually be triggered by the same molecular events, further strengthen this association. Therefore, elucidating the underlying cellular and molecular mechanisms that gather together the two processes is mandatory in order to understand their synergistic effect, and to develop new therapeutic approaches for the management of these disorders that cause a great deal of discomfort, disability, and in some cases death.


AngiogenesisCytokinesDiseaseGrowth factorsInflammationTherapeutic strategies


The inflammatory process

Inflammation is a complex process that requires distinct cell types and factors, which act in a coordinated manner to control tissue damage against pathogenic, traumatic, or toxic injury. The inflammatory process is highly coordinated by pro- and anti-inflammatory molecules that regulate cell chemotaxis, migration, and proliferation [13]. Generally, inflammation ends up in a healing process. However, if this process is not properly ordered, the result is persistent inflammation. Chronic inflammatory conditions have been found to mediate a wide variety of diseases including psoriasis, rheumatoid arthritis, ostheoarthritis, metabolic syndrome-associated disorders, ocular disorders, Crohn’s disease and cancer [4, 512].

Cells and cytokines mediating inflammation

Inflammation encompasses a complex network of chemical signals and cell interactions, which initiates and sustains a host response to tissue damage. Among the cells recruited during this process, leukocytes, as neutrophils and eosinophils, are first recruited upon stimulation of heparin-binding chemotactic cytokines, activating thus specific receptors [2, 3]. Mast cells also play a significant role in the early steps of the inflammatory process, by releasing cytokines, histamine, proteases, and eicosanoids [4]. These mediators, inturn, fulfill many roles, causing vasodilatation and fluid extravasation, promoting endothelium adhesion and further recruitment of inflammatory cells [13]. A network of pro-inflammatory cytokines and chemokines, including members of the interleukin (IL) family, tumor necrosis factor-α (TNF-α) and interferon (IFN) are extensively produced by several cell types at inflamed sites, binding and activating G-protein coupled receptors on immune cells [2, 3]. Activation of these receptors stimulates signaling transduction cascades within the cells, inducing changes in cell morphology and migration. Afterwards, monocytes migrate to the inflamed areas, guided by chemotactic signals [14], where they differentiate into dendritic cells (DC) and into macrophages. The latter will further provide growth factors and cytokines that will engage a wide variety of responses in many distinct cell types, including endothelial cells (ECs), epithelial cells and cells of mesenchymal origin at the local inflamed environment. Upon injury, macrophages and mast cells release cytokines that result in tissue remodeling, and recruitment of additional leukocytes. DC then capture antigens, maturate and migrate to lymph nodes, stimulating hence, adaptive immune cells. Maturation of DC is further enhanced by natural killer cells, which play crucial roles in this interplay between innate and adaptive immunity. In response to DC, B lymphocytes, CD4+ helper and CD8+ cytotoxic T lymphocytes are thus committed to a specific antigen, and will clonally expand in order to enlarge the immune response [15]. Inflammation is regulated by the balance between pro-inflammatory and anti-inflammatory factors that co-exist in injured sites. The switch from a potent inflammatory response toward the healing process is critical, being promoted by local anti-inflammatory signals that are redundant, rapid, and reversible [16]. Whenever pro-inflammatory molecules exceed anti-inflammatory ones, inflammation becomes exacerbated. This imbalance results in the increased production of proteases, proteoglycans, lipid mediators, and prostaglandins that concomitantly enhance the process [17, 18]. As inflammation involves the migration and extravasation of immune cells through the microvasculature, a major role for endothelium has been proposed. In fact, accompanying immune cell migration, inflamed tissues also present increased vascularization.

The angiogenic process

Blood vessel capillaries are lined by a thin layer of ECs and a sheet of extracellular matrix (ECM), called the intima. Veins and predominantly arteries are also composed by contractile smooth muscle cells, the media. Large vessels also have a supportive connective tissue, the adventitia [19].

Angiogenesis is recognized as the growth and remodeling process by which an initial vascular system is modified to form a complex branching network, characteristic of matured vasculature [20]. This is a complex multistep process involving ECM degradation, proliferation, survival, migration, and anastomosis (reviewed by [21]). First, the release of ECM proteases leads to the localized degradation of blood vessels basement membrane. Local ECs change shape, invade stroma, and proliferate, forming tubular structures that join together. This is accomplished by a huge number of anti- and pro-angiogenic factors that function coordinately and synergistically to develop functional blood vessels [20, 21]. Nevertheless, angiogenesis may also be regarded as a harmful process. This is caused by the maintenance of an angiogenic imbalance for long periods, which often accompanies inflammation [5, 20, 22].

Angiogenic sprouting is the principal and most well-described process, involving the proliferation and migration of ECs into avascular regions. But several other angiogenic mechanisms have also been proposed [23]. Accumulating evidence indicates that the recruitment, incorporation, and in situ differentiation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) can also efficiently contribute to angiogenesis in different pathophysiological settings, by a process named adult vasculogenesis [2426].

Coordination of angiogenesis is accomplished by a variety of activators and inhibitors that sequentially synchronize the complex series of events [21]. This process requires the activation of many receptors by numerous ligands, including the placental growth factor (PlGF), acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), angiopoietins (mostly Ang-1 and -2), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and platelet derived-endothelial cell growth factor (PD-ECGF), among many others [2731]. Despite the increasing number of factors playing a role in angiogenesis, the vascular endothelial growth factor (VEGF) signaling on ECs represents the major rate-limiting step during the process [32]. VEGF acts by engaging with its tyrosine kinase receptors VEGFR-1 and VEGFR-2 in ECs. Although VEGF binds both receptors, it appears that most of its biological functions are mediated by VEGFR-2 signaling, thus inducing migration, survival, and proliferation of pre-existing ECs and the formation of a new vasculature [3335]. In addition, VEGF is also the major factor involved in EPCs mobilization from the BM to the peripheral circulation and to angiogenic sites, where they differentiate and integrate the neovasculature ([36], reviewed by [37]).

The aforementioned angiogenic factors are up-regulated by several stimuli, including steroid hormones, increased blood pressure, or shear stress [5, 2022, 3840]. Cyclo-oxygenase-2 (COX-2) is an inducible enzyme responsible for the increase in prostaglandin biosynthesis during inflammation. This enzyme also contributes to increased angiogenesis by up-regulating VEGF production [41], indicating therefore that inflammation might be one of the modulators of pathological angiogenesis.

The molecular links between chronic inflammation and angiogenesis

Chronic inflammation and angiogenesis are, thus, two processes that assemble together. Hypoxia is a common stimulus for both these processes, resulting in the accumulation of macrophages and other immune cells [42], as well as, in increased production of growth factors [2022, 27, 32, 43]. A huge amount of evidence indicates that many of the cells that play a role during inflammatory processes release several factors that act directly or indirectly on vascular ECs [1, 4, 11, 13, 14, 18, 21, 22]. Alternatively, angiogenesis sustains inflammation, by providing oxygen and nutrients for the metabolic needs of the cells present at inflammatory sites. Figure 1 illustrates this complex interplay between chronic inflammation and angiogenesis. This is accomplished in the first place by the production of nitric oxide (NO), a well-established inflammatory agent produced by the activation of inducible NO Synthase (iNOS) in inflammatory cells. NO stimulates vessel dilation and permeability, a feature required for immune cell extravasation (Fig. 1) (reviewed by [3]). But many other pro-inflammatory cytokines and chemokines released during inflammation are potent activators of neighboring ECs to attract blood-derived inflammatory cells (Fig. 1). In fact, one of the most crucial steps involved in this entire process is the adhesion of immune cells to the endothelium, which embraces cell rolling, tethering, and firm adhesion to vascular wall ECs [44]. Adhesion molecules play a relevant role in this stage of the inflammatory process, and are regulated by a variety of pro-inflammatory mediators released by different cell types [44, 45]. E-selectin, one of these adhesion molecules, is practically absent in matured vessels, but highly expressed in activated angiogenic ECs, participating in immune cell extravasation [46]. Identical roles were described for intracellular and vascular cellular adhesion molecules, ICAM-1 and VCAM-1, respectively, expressed at the endothelial surface. These receptors engage specific counter-receptors on immune cells, promoting their adhesion to ECs [45]. Integrins, a family of cell-adhesion receptors composed by α and β subunits, also mediate inflammatory cell-adhesion to the ECM. αvβ3 integrin, usually absent from the quiescent vasculature, is thoroughly expressed at activated endothelium in inflammatory conditions [4547]. On the other hand, inflammatory mediators produced by immune cells target fibroblasts and ECs to release angiogenic factors [1, 48], providing indirect stimuli for angiogenesis to occur. Conversely, inflammatory cells can themselves directly release angiogenic factors, such as VEGF, Ang, bFGF, HGF, PDGF, transforming growth factor-β (TGF-β), TNF-α, among many others, at inflammatory loci, which exert mitogenic and migratory effects in endothelium [4951]. Hypoxia is one characteristic feature of inflammation, inducing the up-regulation of hypoxia-inducible factors (HIFs) expression, which in turn promotes transcription of several angiogenic genes, including VEGF and Ang-2 [4, 48].
Fig. 1

Schematic diagram illustrating the cross-talk between chronic inflammation and angiogenesis. Inflammatory cells secrete growth factors, proteases and cytokines that induce ECM degradation, EC growth, migration and invasiveness capacity, promoting, hence, angiogenesis. Conversely, vascular wall cells enhance chronic inflammation providing cytokines, oxygen and nutrients, and enabling inflammatory cells chemotaxis. This loop further enhances both inflammation and angiogenesis. ECM, extracellular matrix; EC, endothelial cells; SMC, smooth muscle cells; NO, nitric oxide

Activation by pro-inflammatory mediators of the nuclear factor kappa-B (NF-κB), a transcription factor, is a primary event in inflammation. NF-κB is a critical molecule regulating the expression and function of a wide spectrum of genes involved in cell survival, growth and migration, including matrix metalloproteinases (MMPs), urokinase type of plasminogen activator (uPA), V-CAM, I-CAM, and E-selectin adhesion molecules [2, 3, 5254]. Many reports associate this factor with inflammation and angiogenesis [55, 56]. Recent findings propose that inflammation and angiogenesis rely both on the cross-talk between NF-κB and Ang-Tie-2 signaling pathway [55, 57]. In addition to its angiogenic role in blood vessel support cells detachment, Ang-2 also up-regulates several pro-inflammatory pathways leading to leukocyte recruitment and infiltration through NF-κB signaling interaction [57]. Altogether, the support of several distinct cell types enables the continuous production of inflammatory and angiogenic molecules, as well as, the activation of MMPs that degrade the ECM [45].

The increasing number of pathologies displaying chronic inflammation and angiogenesis renders this subject a mainstay that should be adequately revised. The current paper provides a review of the information on this interplay and its relevance in the development and progression of many disorders. The identification of the molecular pathways involved will be of potential benefit to design novel therapeutic strategies.

A close association between chronic inflammation and angiogenesis in lymphatics has also been reported to play a role in several disorders. This has previously been reviewed [58]) and is therefore beyond the scope of the present review.


Psoriasis is a chronic inflammatory dermatosis, which affects approximately 2% of the population in the western world. It is characterized by the presence of erythematous lesions covered by silver-white plaques, which are frequently located symmetrically on the extensor surfaces of the limbs (elbows and knees), scalp, and intergluteal cleft [59, 60]. Psoriasis possesses a strong genetic component, but it also relies on exogenous environmental stimuli, including mechanical trauma, stress, and infection [5961]. This disease is defined by an extensive inflammatory response (involving mainly T cells, macrophages, and dendritic cells), accompanied by subsequent proliferation and differentiation of cells from the skin, namely epidermal keratinocytes, and expansion and dilation of superficial dermal microvasculature.

In contrast to normal skin, where blood vessels arise in the upper horizontal dermal vascular plexus, psoriatic microvasculature is restricted to a superficial thin layer of ECs and basement membrane, forming tortuous and leaky angiogenic vessels that facilitate leukocyte migration into injured skin. Psoriatic skin contains, thus, high numbers of DC, which produce iNOS together with several other pro-inflammatory cytokines [62]. Altogether, these stimulate inflammation, vessel dilation, and permeability.

Molecular inflammatory and angiogenic cross-talk

At the molecular level psoriatic phenotype is characterized by the presence of endothelial E-selectin and of an abnormal pattern of integrins such as αvβ3 integrin, which brings together angiogenesis and inflammation [6365]. Accordingly, T lymphocytes activated by antigen-presenting cells, release T helper type 1 (Th1) cytokines and TNF-α, which promote changes in keratinocytes [59]. These, in turn, secrete a huge amount of chemotactic factors that target both polymorphonuclear cells and ECs, including bFGF, Ang-2, and VEGF [65, 66]. In addition, increased VEGF serum levels have also been described in psoriatic patients [67]. Microvasculature in psoriatic lesions often express VEGFR-1 and VEGFR-2 [68], suggesting that keratinocyte-derived VEGF is a potent mitogen for dermal microvascular ECs in psoriasis. Moreover, a down-regulation of anti-angiogenic factors, as thrombospondin (TSP), has also been reported [69]. The significant association between the inflammatory factor TNF-α and the angiogenic-associated MMP-9 in sera from psoriatic patients reinforced the assumption that these two processes are clustered together in psoriasis [70]. Transgenic animal models also contributed to identify the pathways involved in psoriatic pathogenesis. VEGF gene transgenic overexpression in the mouse skin resulted in vascular hyperplasia, increased T cell infiltration and epidermal hyperplasia, resembling a psoriatic-like phenotype [71]. These features imply that VEGF, besides stimulating angiogenesis, can also trigger unexpected immune responses, suggesting the existence of a complex coordination between blood vessels, keratinocytes, and immune cells.

Therapeutic management

Psoriasis is a slow developing disease that rarely remits spontaneously. Current treatment comprises hydration, UV light, and a few pharmacological agents [59]. Additionally, some therapeutic agents for psoriatic lesion treatment already target chronic inflammation and angiogenesis. Vitamin D and retinoic acid are two of these agents that effectively decrease angiogenesis [59]. Cyclosporine A (Cy A) presents anti-psoriatic activity by preventing cytokine secretion. Recent reports indicate that it also down-regulates VEGF-induced angiogenesis through COX-2 mediation [72]. Monoclonal antibodies (mAb) targeting TNF-α a cytokine with inflammatory and angiogenic effects were beneficial in psoriatic skin, resulting in a substantial decrease in T cells. Antagonists of other cytokines overexpressed in psoriasis with established roles on both processes are also being tested [73, 74]. Despite a few therapeutic agents used in psoriasis targeting the pathways that gather inflammation and angiogenesis together, a careful appreciation of the involved mechanisms must be provided. It would be better to clearly define the complex interaction between chronic inflammation and angiogenesis in psoriatic lesions prior to developing future therapeutic approaches.

Rheumathoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease of unknown etiology with a prevalence of roughly 1% worldwide [75]. RA normally presents as symmetric arthritis, causing typical deformities of the synovial-lined joints of the hands and feet, ankles, knees, wrists, elbows and shoulders, causing increased morbidity and mortality [75]. The synovium lines the non-cartilagineous surface in joints and normally consists of two or three layers that provide nutrients to the adjacent avascular cartilage and produces a viscous, acellular fluid that fills the joint cavity enabling it to move smoothly [76]. Normal synovium contains sprinkled blood vessels and a limited amount of macrophages, synoviocytes, fat cells, and fibroblasts [77]. This synovial architecture changes drastically in RA patients. RA is primarily characterized by a pre-vascular highly inflammatory phase, followed by a vascular phase with strong increase in vessel growth. In the pre-vascular phase, the synovium in RA becomes inflamed and increases greatly in mass, due to hyperplasia of the lining cells. Blood-derived cells, including lymphocytes and macrophages infiltrate the sublining of the synovium, leading to local invasion at the synovial interface with cartilage and bone. This results in an invasive and destructive front, called pannus [78]. The development of the pannus is characterized by an increased density of sub-lining capillaries and post-capillary venules, supporting the progression of arthritis and underlying the important role of the vasculature in this invasive and destructive inflammatory joint disease [79].

The increased number of blood vessels correlates with synovial cells hyperplasia and mononuclear cells infiltration [80]. This raise in synovial cell proliferation and fluid volume induces hypoxia, causing a metabolic demand for oxygen and nutrients that in turn triggers angiogenesis [22, 81, 82].

Inflammation and angiogenesis interaction

A network of pro-inflammatory cytokines and chemokines, such as several interleukins (as IL-1, IL-6, and IL-15), IFN, TNF-α and TNF-like weak inducer of apoptosis (TWEAK), are highly produced by several cell types at the inflamed synovium. These induce the ongoing recruitment of immune cells, resulting in the up-regulation of adhesion molecules on the synovial endothelium and allowing their migration into the joint [8386]. Additionally, these infiltrated inflammatory cells also synthesize pro-inflammatory cytokines at the rheumatoid synovium, regulating both chronic inflammation and angiogenesis [87, 88]. In fact, some of these pro-inflammatory mediators can directly and/or indirectly stimulate the angiogenic process, by up-regulation of vascular endothelial mitogens and promotion of endothelial anti-apoptotic signaling pathways, as the phosphatidylinositol 3-kinase (PI3-K)/Akt-1, COX-2 and NF-κB, which promote angiogenesis and synovial inflammation [8994]. The expression of both Ang-1 and Ang-2 was observed in chronic inflamed synovium, being Ang-2 expressed at remarkably higher levels [95, 96]. The finding that Ang-2 plays inflammatory roles in ECs further emphasizes its dual role in inflammation and angiogenesis [57, 97].

Vascular endothelial growth factor levels were found to be markedly higher in the serum and synovial fluids of patients with RA than in healthy controls [98, 99], being also highly expressed by several cell types in the RA synovium, eventually by the action of pro-inflammatory cytokines [89, 100102]. The presence of the VEGF receptors (VEGFR-1 and VEGFR-2) in microvascular ECs reinforces the important paracrine interaction in the RA-associated angiogenesis [103, 104]. The up-regulation of VEGF in RA synovial tissue also stimulates EPCs and leukocyte recruitment [25, 26, 36]. Besides, VEGF expressed at the inflamed synovium may stimulate the production of MMPs, promoting ECM degradation within the joints [105, 106].

Current and future therapeutic strategies

Initial therapeutic advances involved the use of non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease modifying anti-rheumatic drugs (DMARD), which are considered the mainstay for the treatment of RA [94, 107]. However, none of these therapeutic interventions is curative and all have significant side-effects. On the other hand, the relevance of cytokines such as TNF-α, IL-1, IL-6, and IL-15 in RA development has led to the investigation and development of innovative biological therapies [108112]. Blocking TNF-α with infliximab (a neutralizing mAb) [113] was beneficial in DMARD-resistant RA patients in multiple clinical trials, resulting in synovial vascular regression. Furthermore, blockade of TWEAK, a putative novel arthriogenic mediator, significantly reduced joint inflammation and synovial angiogenesis [86]. Besides biological therapies, recent research has focused on studying the target effects of signaling transduction pathways activated in RA, as the PI3-K, involved in the activation of endothelial anti-apoptotic signals and in the regulation of immune cell transport into inflammatory sites [114].

Since the rheumatoid pannus relies on the development of new vasculature to sustain growth, it seems likely that suppression of new blood vessel formation would delay arthritis progression. Preventing angiogenesis would minimize extravasation of immune cells and abrogate pro-inflammatory cytokines. Accordingly, non-specific anti-angiogenic compounds, as TNP-470, taxol, and thalidomide, have been shown to inhibit neovascularization and pannus formation in animal models of arthritis [115118]. The fumagillin derivatives AGM-1470 or TNP-470 inhibit both angiogenesis and arthritis in rodents, and sulfasalazine, commonly used to treat a variety of diseases including RA, reduces endothelial proliferation and chemotaxis [119]. VEGF signaling has also been addressed in murine models of arthritis using a soluble form of the VEGFR-1 (sFlt-1) [120] and anti-VEGF polyclonal antibodies [121, 122]. VEGFR-1 may be a key player in RA, due to its effects on BM hematopoietic cells and immunity of monocyte/macrophages [123]. Although these pre-clinical therapies seem valuable, the whole molecular basis of the VEGF/VEGFRs system actions is not fully clarified. Further studies in order to accurately evaluate the beneficial outcomes of the suppression of these molecules in RA patients are required. Additional studies invested also on alternative angiogenesis target approaches in arthritis, including endostatin [124], TSP-1 and -2 [125], as well as, αvβ3 integrin [126128]. Despite all these efforts to target inflammation and angiogenesis in RA, and at least partly due to the adverse side-effects of some of these drugs, until now no clinical approval has been obtained for arthropathy patient treatment [121123, 129]. In theory, anti-angiogenic treatment alone or combined with anti-TNF-α in RA would be beneficial without augmenting potential adverse effects.


Osteoarthritis (OA), also known as degenerative, hypertrophic, or age-related arthritis, is the most common type of arthritis, comprised by a group of disabling chronic painful conditions affecting synovial joints [130]. Osteoarthritis develops very gradually and usually affects one or a few joints, particularly of the hips, knees, lower lumbar and cervical vertebrae, fingers and hands, and is present in 63–85% of individuals over 65 years of age. The etiology of OA is multifactorial and includes both systemic and local biomechanical factors. OA is considered a degenerative disease and commonly described as a non-inflammatory disease in order to distinguish it from “inflammatory arthritis”, such as RA [6]. However, inflammation is increasingly recognized to contribute to the progression of OA [131136]. OA synovial membranes present inflammatory infiltrates, primarily consisting of T cells and monocytes, in over 50% of patient samples [133]. Serological evidence of inflammation has also been reported in OA, since elevated levels of C-Reactive Protein (CRP) have been detected in OA patient’s sera and correlated with disease progression [135]. Furthermore, several pro-inflammatory cytokines and chemokines, including IFN, IL-1, IL-6, IL-8, IL-12, IL-17, TNF-α and stromal derived factor-1 (SDF-1) have been identified on synovial infiltrates in OA [131, 133, 137]. At the OA synovium these molecules activate specific receptors present on ECs, cartilage, and bone cells, stimulating them to produce MMPs and plasminogen activator, which impair the structure and function of the cartilage by reducing its repairing capacity [76, 138]. Increased release of COX-2 in subchondral osteoblasts is thought to play a role in stimulating bone resorption [139, 140]. Furthermore, the abovementioned mediators may additionally enhance neovascularization at the osteoarthritic synovial tissue [141, 142].

In summary, inflammatory cells that are abundant in the inflamed osteoarthritic synovium can induce angiogenesis directly by producing angiogenic factors, and/or by secreting cytokines that indirectly stimulate ECs and fibroblasts to release VEGF at the OA synovium [143, 144]. In addition, VEGF expression also contributes to both neovascularization and inflammation, by inducing the mobilization of immune cells [145]. The expression of angiogenic factors is further enhanced by the hypoxic conditions that are characteristic in OA [146, 147]. Although there is little information regarding other neovascular mediators, VEGF signaling seems to be the crucial molecular mechanism, exacerbating both osteoarthritic angiogenesis and inflammation at the synovial endothelium.

An overview of the therapeutic approaches

Treatment of OA is intended to controlling pain, maintaining function, minimizing complications, and slowing down the development of the disease. These therapeutic goals can be accomplished by non-pharmacological measures, such as termal modalities and exercises, together with the use of analgesics, NSAIDs, and more recently slow acting drugs in osteoarthritis (SADOAs) [148]. The identification of inflammation as a common feature in OA renders anti-inflammatory agents promising approaches, but their application in osteoarthritic patients OA remains in its infancy [149]. Since the neovascular process has been ascribed to contribute for OA, targeting angiogenesis would probably be beneficial in delaying the course of disease as well.

In contrast to the use of angiogenesis blockade agents for RA treatment, there are still no pre-clinical investigations regarding angiogenesis inhibition in OA. This is mainly due to the recent recognition of the angiogenic process as an important event in the pathogenesis of OA.

Crohn’s disease

Crohn’s disease (CD) is a chronic inflammatory bowel disease that affects one in 250 people in northern Europe. Despite the low mortality, CD’s morbidity is substantial [150]. CD involves damage in the intestine epithelial tissue, in which severe and continuous inflammatory conditions disturb the intrinsic epithelium repair system, resulting in refractory intestinal ulcers (reviewed by [151]). The dominant clinical features are recurrent episodes of diarrhea, crampy abdominal pain, and fever lasting days to weeks. Malabsorption can develop, and being CD a multisystemic disease, extraintestinal inflammatory manifestations can also occur. Although the etiology and mechanisms of tissue damage in CD remain to be completely identified, an abnormal immune reactivity is ultimately responsible for damaging the gut and causing clinical manifestations [150, 152]. As CD involves inflammation, ulceration, and regeneration of the intestinal mucosa during the course of the disease, it is likely that the angiogenesis counterpart might also be fundamental in CD pathology. However, whether such an angiogenic process is simply a secondary event triggered in the context of a reparative process, or whether causative links exist between pathological angiogenesis and CD is under investigation [153].

The breakthrough that CD is genetically associated with mutations of the NOD2/CARD15 gene, whose product is a bacteria-recognizing cytoplasmic protein in monocytes, points to defective mechanisms of bacterial sensing as the link between the gut flora and the altered immune response found in CD [154]. CD does predominantly involve a Th1 response. CD mucosal T-cells are resistant to apoptosis and divide rapidly, increasing the inflammatory status [155]. In addition, a raise in the synthesis of other pro-inflammatory cytokines was found, accompanying the influx of other inflammatory cells into the mucosa [156158]. These molecules contribute to tissue damage either directly or indirectly by enhancing the production of MMPs and growth factors, which conduce both to ulceration and mucosal repair, also enhancing new vessel formation.

Angiogenesis and inflammation

Increasing evidence demonstrates that angiogenesis and inflammation are associated with the development of CD. Blood vessel growth is inherent to inflammation and is associated with structural changes that result in an expansion of the tissue microvascular bed [5, 159]. Lately, CD has been reported to be associated with excessive angiogenesis [160]. However, detailed information about the angiogenic status and the molecular pathways activated in the inflamed mucosa is rare and controversial [161]. Vascularization in CD was particularly observed in areas of active inflammation, reinforcing the co-dependence of these two processes in chronic inflammatory disorders [162]. Moreover, the microvessels show an enhanced leukocyte adherence by increasing I-CAM and E-selectin expression and an inappropriate preferential recruitment of naïve monocytes and T cells to the chronically inflamed intestine, compared with non-pathological bowel areas [161]. A restricted expression of chemokines in the mucosal surface of the gut may explain the selective leukocyte trafficking to the inflamed area. The presence of αvβ3 integrin, characteristic of angiogenic vessels, was increasingly expressed on CD mucosal-associated microvessels. There is still a great controversy regarding the presence of vascular growth factors in CD inflamed intestinal mucosa [153, 162]. However, a microvascular EC dysfunction might occur, due to a loss of NO-dependent dilation, conducing to poor wound healing and maintenance of the inflammatory condition of the intestinal mucosa through a reduced leukocyte migration [163].

Available and prospective therapies

Conventional therapy for CD consists of sulfasalazine agents, corticosteroids [164] and chemotherapeutical drugs, such as azathioprine and methotrexate, aimed at inducing immunosupression and inflammation blockade [165167]. The use in clinical studies of thalidomide, known to downregulate TNF-α and IL-12 production and to act as an angiogenic inhibitor, was also reported [168]. However, the use of these agents has several adverse effects that significantly hamper the clinical management of this disease (reviewed by [167]). The development of agents against TNF-α, IL-12, INF, and α4β7 integrin, has shown to induce good tolerance and safety, providing promising clinical outcome in CD patients [169171]. However, future research is required, since inhibition of each of these pro-inflammatory mediators displayed rapid loss of response in lasting therapy [171].


Cancer is a multistep process during which cells acquire genetic alterations that drive the progressive transformation of normal cells into highly malignant cells. Cancer cells are characterized by uncontrolled growth, escape from apoptosis, sustained angiogenesis, tissue invasion, and metastasis [172]. Chronic inflammation has also been implicated in cancer development and may influence many of the aforementioned processes that contribute to the multistage development of tumors [173, 174]. Accordingly, in 1863, Rudolph Virchow first discovered leukocytes in neoplastic tissues, suggesting a link between inflammation and cancer (reviewed by [175]). Currently, it has been demonstrated that the tumor microenvironment highly resembles an inflammatory site. These environmental changes favor tumor progression by the secretion of cytokines and chemokines by leukocytes, generating reactive oxygen and nitrogen species that can directly damage cell cycle controlling genes [176].

Corroborating this assumption, population-based studies reveal that individuals who are prone to chronic inflammatory diseases have an increased risk of cancer development [174]. In addition, mutations or polymorphisms in genes encoding immune modifiers (such as IL-1 and TNF-α) exist in individuals with chronic inflammatory disorders presenting an increased incidence of cancer [174, 177, 178]. Most remarkably, inhibition of chronic inflammation in patients predisposed to cancer development or presenting pre-malignant disease results in chemoprevention. These studies revealed that long-term usage of anti-inflammatory drugs, selective COX-2 inhibitors, significantly reduces cancer risk [179].

Inflammation and angiogenesis

Angiogenesis is crucial for tumor development and progression [21, 23]. Several clinical studies have reported the abundance of immune cells in human tumor samples and correlated their presence with increased angiogenesis or clinical outcome [174, 180, 181]. Tumor microenvironments are rich in immune cell-derived factors with pro-angiogenic effects such as IL-1, IL-6, IL-8, TNF-α, COX-2, and VEGF [174, 182], with a concomitant raise in neovascularization [183]. Among these, TNF-α is the best characterized inflammatory mediator in carcinogenesis so far. TNF-α induces activation of NF-κB [184], which is involved in the regulation of both inflammation and angiogenesis [3]. VEGF production by tumors and/or tumor-infiltrating leukocytes contribute to increased angiogenesis and EPC recruitment, fostering tumor development [185, 186]. Recently, it was also demonstrated that besides EPCs, additional BM-derived cells, mainly of myelomonocytic origin, are also co-mobilized, by VEGF action, to tumors [25, 26]. These myelomonocytic cells localize to tumor perivascular sites, stabilizing tumor neovessels and contributing to the angiogenic process in early phases of malignant development [25, 26].

This inflammation–angiogenic connection is further strengthened by the enhanced expression of COX-2 in several tumor types where it up-regulates angiogenic factors, as well as, pro-inflammatory cytokines [37, 187]. Overall, there is compelling evidence for a strong link between chronic inflammation and angiogenesis in cancer settings.

Anti-angiogenic strategies

The discovery that tumor development and progression depend on angiogenesis led to high expectations that blocking this process would be of therapeutic benefit. It took over three decades and numerous pre-clinical studies using many anti-angiogenic approaches to clearly demonstrate the usefulness of anti-angiogenic agents [188, 189]. Many distinct strategies have been used with varying degrees of success in human-cancer clinical trials. Due to its biological importance, targeting VEGF signaling is the most investigated approach [190]. In fact, the first anti-angiogenic agent, an anti-VEGF mAb, was approved in combination with chemotherapy for the treatment of patients with metastatic colorectal cancer [191]. Furthermore, anti-inflammatory drugs were shown to display anti-angiogenic effects and their use might also be beneficial for neoplastic therapy [192].

Metabolic syndrome

Metabolic syndrome (MS) is a highly prevalent condition in the western world, representing a cluster of several risk factors for cardiovascular disease, namely visceral obesity, dyslipidemia, insulin resistance, and hypertension, all of them being major causes of morbidity and death. Increasing evidence confirms that chronic inflammation is a predominant feature in MS [193]. Although many pro-inflammatory cytokines have been reported to play a role in MS, the associated mechanisms are not yet clear. Accordingly, IL-6, resistin, TNF-α, fibrinogen and CRP are released by several cell types into the circulation, stimulating then hepatic CRP production [194]. CRP, in turn, further enhances insulin resistance, providing a systemic pro-inflammatory stimulus [195]. MS is also regulated by the receptor of very low-density lipoproteins (VLDL). This was highlighted by the fact that mice lacking VLDL receptor (VLDLR −/−), presented decreased adipose tissue mass and lack of insulin resistance [196]. In addition to its specific ligands, apolipoprotein (Apo)E and lipoprotein lipase (LPL), VLDLR also binds to TSP-1, urokinase plasminogen activator/PAI-1 complex, protein–serpin complexes, and tissue factor pathway inhibitor (TFPI) [197200], implying a putative role of VLDLR in angiogenesis as well. Moreover, the LDL particles become oxidized, promoting thus inflammation. Inversely, the low levels of the anti-oxidant HDL particle in the MS blunt another anti-inflammatory mechanism. Thus, all the pathological situations associated with MS share inflammation and angiogenesis [201]. The interaction of these processes will be discussed next in obesity, diabetes, and atherosclerosis, three of the most relevant MS-associated disorders.


Lately, obesity has emerged as a major public health problem due to its high prevalence and association with increased risk for development of type 2 diabetes mellitus and cardiovascular disease, two highly incident disorders in western world [193]. The adipose tissue has been primarily viewed as a fatty acid storage deposit. However, more than an inactive bystander, the recent finding that adipocytes release leptin and adiponectin hormones, implies that this fat-storage amorphous tissue is actually a dynamic and metabolically active one [202]. In fact, adipose tissue also synthesize and release a huge number of signaling proteins, the adipokines, which are involved in regulating many cellular processes, including inflammation and angiogenesis [2, 203]. Large-size adipocytes mainly in visceral adipose tissue are prone to rupture, therefore evoking an inflammatory reaction [202]. As a matter of fact, macrophages present in adipose tissue around dead adipocytes constitute a source of inflammatory factors [203]. Accordingly, obesity is now being considered as a state of chronic inflammation, as pro-inflammatory cytokines are increased in the plasma of obese patients [203].

Adipose tissue is one of the few adult tissues that has the ability to grow and regress. This characteristic strongly depends on active angiogenesis, since the expansion of capillary beds is necessary for tissue growth [204]. In agreement, adipose tissue contains extensive capillary networks surrounding adipocytes, which are probably induced by adipocyte-secreted growth factors and hormones [203]. In accordance, increased levels of these adipokines with pro-angiogenic activity have been described in overweight and obese patients [205]. It was also demonstrated that blocking VEGF signaling resulted in decreased angiogenesis in pre-adipocyte differentiation and a reduction in adipose tissue mass as well, leading to the assumption that adipogenesis and angiogenesis are reciprocally regulated [206].

Therapeutic options and perspectives

Treatment of obesity was shown to be of utmost difficulty. Obesity development depends on a huge number of factors and its prevention/treatment likewise demands a multitude of interventions. Pharmacotherapy has been limited to a few drugs that control abnormal fat accumulation mostly by acting in brain receptors, inhibiting appetite and energy balance pathways [207, 208]. However, this short-term beneficial drugs have substantial side-effects and after treatment cessation, there is a rebound weight gain. Given the large number of adipokines that present pro-inflammatory effects [206], together with the recent concept that adipose tissue mass strictly depends on angiogenesis, new targets are now emerging, namely peptides targeting prohibitin, a vascular marker of adipose tissue [209].


A characteristic feature of obese patients is the development of insulin resistance and thereafter type 2 diabetes mellitus. Due to its increasing incidence, diabetes will probably be a leading cause of morbidity and mortality in the near future. This is another pathological situation carrying together inflammation and angiogenesis. Diabetes mellitus also exhibit enhanced angiogenesis leading to diabetic retinopathy and potential disruption of atherosclerotic plaques [210]. Conversely, defective angiogenesis is observed elsewhere, leading to wound healing impairment, which is also characteristic among these patients. Diabetics also present a reduction in the number of circulating EPCs [211]. Following activation of the VEGF signaling pathway, vessels become leaky and angiogenic in the whole body. In the retina, this leads to local inflammatory response resulting in vascular sprouting [212]. These new vessels easily disrupt, leading to vitreous hemorrhage and retinal detachment (see ocular disorders). Similarly, proliferation of the arterial wall may result in sprouting, plaque destabilization, and rupture with concomitant thrombosis (see atherosclerosis). Afterwards, lack of VEGF signaling in endothelium and in monocytes prevents arteriogenic response, which is further reduced by the absence of EPCs. It is very likely that this complex picture of diabetes involves many other unknown factors. For example, the activation of other circulating angiogenic growth factors, such as FGF, HGF, PlGF, and PDGF can also be abrogated by glycation [213], a common feature occurring in diabetes. Also, glycation end-products in persistent hyperglycemia in diabetes further trigger arterial inflammation [214].

Therapeutic perspectives

Recent efforts have been made to identify new pharmacological therapies for the management of diabetes. These include peroxisome proliferator-activated receptor (PPAR) agonists or insulin analogs, incretin agonists or glucagon-like peptide-1 analogs and cannabinoid receptor antagonists [215]. These agents have been designed against appropriate metabolic targets. Knowing that inflammation and angiogenesis processes are active in diabetes, led to the search of new pharmacological compounds that will probably become available within the next decade. Despite all this relevant knowledge regarding the angiogenic pathways that are mobilized in diabetes, only few trials targeting angiogenesis have been reported [216]. For instances, a clinical trial using PDGF in diabetic patients showed a significant improvement in chronic neurotrophic diabetic foot ulcers healing [217]. Furthermore, anti-angiogenic drugs are being used for diabetic retinopathy treatment with promising results (see ocular disorders). This is probably due to the complex synergism between inflammation and angiogenesis activated pathways in diabetes.


Atherosclerosis is characterized by EC dysfunction and damage, and smooth muscle cell (SMC) proliferation and migration [218]. This blood vessel deregulation initiates inflammation by infiltration and retention of low-density lipoproteins (LDL) in the intima within the artery wall. Oxidation of LDL or enzymatic attack in the intima leads to phospholipid release, which further contributes to activate ECs [219]. This process is further enhanced by hemodynamic flow profiles, which induce the up-regulation of adhesion molecules and inflammatory genes by ECs [218]. Adhesion of leukocytes to ECs leads to cytokine release in the underlying intima and stimulates their migration into the subendothelial space. In addition, activated immune cells in the plaque produce pro-inflammatory mediators, which are all established pro-angiogenic factors as well [218, 219]. Superficial erosion or desquamation of ECs that form the monolayer covering the intima is very frequent, providing a nidus for a platelet thrombus. IFN secreted by T lymphocytes prevents collagen synthesis by SMC, destabilizing the atherosclerotic plaque. In addition, several cytokines released in the atheroma, such as IL-1β, TNF-α and CD40 ligand, increase MMP expression in mononuclear cells, EC and SMC [218, 220]. Platelets are also key players in EC activation. They synthesize glycoproteins Ib and IIb/IIIa that bind EC surface molecules [220]. Moreover, adherent platelets express chemokines, as monocyte chemoattractant protein-1 (MCP-1) and SDF-1, leading to recruitment of EPCs involved in neovascularization and plaque progression. Platelets act as inhibitors of angiogenesis rather than activators, since they release specific platelet factors, which strongly inhibit angiogenesis [220]. However, macrophages residing in the plaque also produce angiogenic factors [219]. The fragile angiogenic microvessels not only serve as sites for hemorrhage and thrombosis, but may also provide oxygen and nutrients required for the plaque to grow. Using apolipoprotein E-deficient mice, which are prone to developing atherosclerotic lesions it was shown that treatment with the anti-angiogenic agent endostatin led to inhibition of plaque growth [221]. Accordingly, the use of angiogenesis inhibitors in animal models of atherosclerosis is promising and seems to be effective.

Therapeutic strategies

Since the inflammatory process plays a crucial role in atherosclerosis, the use of anti-inflammatory agents appears to be a very attractive approach. One of the potential targets is COX-2. However, inhibition of COX-2 and synthesis of eicosanoids in atherosclerotic patients resulted in increased incidence of cardiovascular disorders [222]. Therefore, a careful understanding of the mechanisms that underlie inflammation in atherosclerosis is primordial. Interestingly, statins, molecules that inhibit cholesterol synthesis, exert anti-inflammatory and anti-angiogenic properties [223]. Despite their anti-inflammatory and anti-angiogenic mechanism having not yet been established, statins prevent prenylation, which is required for activation of many protein receptors present at the cell membrane. Both inflammation and angiogenesis involve an extensive number of paracrine interactions with activation of many transduction pathways through specific prenylated receptors at the cell membrane. The inactivation of these receptors by statins might be beneficial against atherosclerosis [224]. Therapeutic strategies directed against proteoglycans are also being tested, since proteoglycans are essential for the activation of platelet-derived cytokine secretion [220]. Given the well established effect of SMC in atherosclerotic plaques, increased apoptosis of these cells would also be a putative target against this pathology. An emergent study shows that intense intimal inflammation is found in arteries in atherosclerotic plaques, due to accumulation of lipids and cell debris as a consequence of SMC-induced apoptosis [225]. Indeed, expression of IL-1α, IL-8, and MCP-1 increase after SMC apoptosis causing extensive infiltration of macrophages [226].

Ocular disorders

Ocular neovascularization (ONV) occurs in several ocular diseases, presenting mainly as retinal neovascularization (RNV) in diabetic retinopathy (DR), and as choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Ocular neovascularization became the first cause of blindness worldwide [227]. In the adults, the eye vascular system is in a quiescent state, and thus certain eye compartments remain avascular, allowing the normal visual function to be preserved. The abnormal growth of new vessels into these compartments will cause disturbance of light transportation. Moreover, these new vessels are very fragile, leaky and susceptible to hemorrhage, leading to further decrease of visual acuity [228]. Furthermore, concomitant edema and exudates formed will decrease the transparency in the cornea and indirectly cause damage of retinal neurons, resulting in vision loss. Among the different tissues in the eye, the retina, choroid, and cornea are the most frequent sites of ocular neovascularization. The mechanisms that lead to ONV are still not well understood; however, hypoxia associated with up-regulation of VEGF, oxidative stress, and inflammation may contribute to the initiation and progression of the disease.

Inflammation and angiogenesis

VEGF seems to play a central role in the progression of pathological ONV. The co-dependence of angiogenesis and inflammation is well demonstrated in experiments where intravitreal injections of VEGF upregulated ECs I-CAM expression, leading to local adhesion of leukocytes, and hence, to vascular permeability, capillary non-perfusion, and monocyte chemotaxis [11]. These leukocytes may in turn secrete VEGF. In fact, activated macrophages can influence the whole angiogenic process, by inducing alterations in the extracellular matrix, proliferation, and migration of ECs. Experiments using macrophage-depleted mice showed that laser-induced choroidal neovascularization size and leakage were reduced, and so did VEGF levels [229]. Furthermore, VEGF was shown to have pro-inflammatory activity. Specific up-regulation of VEGF was in fact accompanied by enhanced leukocyte adhesion and contributing to pathological ONV [11]. The Ang signaling pathway, which has pro-angiogenic and pro-inflammatory properties, also seems to take part in human choroidal neovascularization [12]. Another solid evidence showing the role of inflammation in the pathogenesis of CNV is that depletion of complement using cobra venom factor inhibited the up-regulation of angiogenic factors and subsequent CNV formation [230]. In C3 complement factor-deficient mice, CNV did not develop, further indicating the role of complement and inflammation in the pathogenesis of CNV [230]. Also, TNF-α, MCP-1, and subsequent leukocyte–endothelial interactions contribute to the blood retinal barrier breakdown and subsequent vascular leakage, as well as, angiogenesis [231].

Treatment of ONV

The classical laser treatment shows some effects on the control of retinal and choroidal neovascularization, but the therapy is destructive to the retina. However, as the mechanisms responsible for the disease are unfolded, new therapies may arrive. Pegaptanib sodium, an anti-angiogenic drug used for CNV, targets VEGF. Pegaptanib proved to be of clinical benefit in phase III AMD trials [232, 233]. Others, like ranibizumab, a murine mAb anti-VEGF, and anecortave acetate, an angiostatic steroid, are being evaluated in clinical trials as well [234]. Clinical trials demonstrated that intravitreal administration of ranibizumab at 4-week dosages prevents visual loss, and seems to improve visual acuity in 30% of AMD patients [235]. Corticosteroids are being studied due to their anti-inflammatory and angiostatic properties. Triamcinolone acetonide downregulates cytokine-induced ICAM-1 expression in ECs and reduces permeability of cultured EC monolayers. Pre-clinical studies have shown that intraocular triamcinolone inhibit laser-induced neovascularization [235]. In a prospective clinical study, a mAb against inflammatory factor TNF-α successfully resulted in regression of CNV and in improvement of visual acuity in patients with exudative AMD [236].

Conclusions: assembling chronic inflammation and angiogenesis toward therapy

Inflammation is crucial to defend our body against pathogens. However, longstanding inflammation results in adverse effects. These, in turn, are further enhanced by the activation of angiogenesis. Several evidences demonstrate that chronic inflammation and angiogenesis can be upheld by the same stimuli. Accordingly, several growth factors and cytokines have been associated with both these processes within the literature (Table 1), further emphasizing the close partnership that gather them together. Strikingly, these two processes seem to depend on each other. Common molecular mechanisms have also been found, corroborating this assumption [57, 97]. It is quite remarkable to realize that a wide range of disorders presenting much distinct etiopathogenic origin carry identical molecular mechanisms that cluster inflammation and angiogenesis together.
Table 1

Literature review of growth factors and cytokines reported to exert both inflammatory and angiogenic roles in several diseases


Pathologic condition

Reference in the literature


Psoriasis, CD

[5860, 154]



[9, 10, 154]


Psoriasis, CD

[5860, 163, 165]


Psoriasis, RA, OA, CD, cancer, MS, obesity, atherosclerosis, OD

[60, 61, 7881, 125, 127, 131, 163, 165, 168, 171, 172, 187, 210, 211, 229]


Psoriasis, diabetes

[60, 61, 206]


Psoriasis, RA, OA, diabetes, OD

[9, 10, 60, 61, 93, 94, 137, 138, 205]


Psoriasis, RA, obesity

[60, 61, 98, 99, 198]





Psoriasis, RA, OA, atherosclerosis

[65, 71, 100, 101, 132, 210, 211]


RA, OA, cancer

[7881, 125, 127, 131, 168, 171, 172]


RA, OA, cancer, MS, obesity, atherosclerosis

[7881, 125, 127, 131, 168, 176, 187, 200, 210, 211]


OA, cancer, atherosclerosis

[125, 127, 131, 168, 176, 218]



[125, 127, 131, 163, 165]






[125, 127, 131]


RA, OA, CD, atherosclerosis

[7881, 125, 127, 131, 163, 165, 211]



[11, 90, 91]

Stromal-derived factor1


[125, 127, 131]

Plasminogen activator


[71, 132]


OA, cancer, atherosclerosis

[133, 134, 173, 214]











MS, atherosclerosis

[194, 211]










Glycoproteins Ib, Iib/IIIa




Atherosclerosis, OD

[212, 222, 223]







CD, Crohn’s disease; OD, ocular diseases; RA, rheumatoid arthritis; OA, osteoarthritis; MS, metabolic syndrome

This emergent knowledge led to the chase of direct-therapeutic approaches against both angiogenesis and chronic inflammation. Targeting new blood vessel formation holds the promise of decreasing influx of immune cells, reducing production of inflammatory and proteolytic mediators, and preventing nutrients supply to an active inflammatory process. Conversely, targeting inflammation would also negatively affect blood vessel formation as described above. However, the intricate interplay between these two processes, discussed in the present paper, blurs the search for therapeutic strategies. Therefore, a careful understanding of the cross-talks between chronic inflammation and angiogenesis must be highlighted for each pathology, aiming at more effective therapies. Nonetheless, as epidemiological and experimental evidence indicates that dietary compounds, such as polyphenols, offer protective effects against both inflammation and angiogenesis [237240], the relevance of natural agents as chemopreventive against disorders associated with chronic inflammation and angiogenesis should also be taken into account, especially because they are easier and cheaper to establish.

However, there is still one question remaining to be answered regarding the interplay between chronic inflammation and angiogenesis: are they cause or consequence?


The authors would like to thank Professor Isabel Azevedo for her helpful discussions, comments and revision of the manuscript. Carla Costa was funded by “Fundação da Ciência e Tecnologia” (SFRH/BPD/20832/2004).

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© Springer Science + Business Media B.V. 2007