ACKNOWLEDGMENTS
Neste pequeno espaço, fica o meu enorme agradecimento e reconhecimento a quem tornou possível este trabalho e/ou fez de mim uma pessoa melhor (se eu me esqueci de alguém, eu prometo que faço um brownie de chocolate para compensar)
Ao meu pai e ao meu irmão, obrigada por me aturarem desde 1988
À Professora Susana Constantino, minha orientadora, um grande obrigado por me ter dado esta oportunidade, por todo o apoio e por todas as loucuras ao longo desta etapa
À Professora Rita Zilhão, que eu tive o privilégio de ter tido como professora durante o 1º ano, o meu obrigado pela disponibilidade constante como orientadora interna nesta fase
À Rita Simões Pereira, obrigada por toda a paciência, por me deixares ser a tua lapa e por todo o apoio moral imprescindível ao longo deste ano.
À Unidade de Angiogénese: Andreia Pena (quando é que escrevemos o livro?), Augusto Ministro (isquemias às 7h?), Paula Oliveira (oláaaa!), Filipa Marques (LCM queen e PCR master!), Adriana Correia (um dia vou ter contigo!), Carolina Cardina (a minha inspiração para o Prezi), Telmo Freitas (ainda estou à espera do tampão de lise) e Liliana Carvalho (bolachinhas!!)
Ao Laboratório de Histologia e Patologia Comparada do Instituto de Medicina Molecular À unidade de BioImagem do Instituto de Medicina Molecular
Ao Departamento de Anatomia da Faculdade de Ciências Médicas da Universidade Nova de Lisboa
À minha cunhadinha: you rock!
À minha família, em especial às minhas priminhas, Alessia e Valentina, por me deixarem ser mais criança do que vocês
Aos meus amigos de sempre e para sempre: Gus, Lili, Rita, Clarinha, Avila, Patini, Dentinettes, obrigada por serem quem são
i LIST OF ABBREVIATIONS
ANG: Angiopoietin ANG-1: Angiopoietin-1 ANG-2: Angiopoietin-2 EC: Endothelial cell ECM: Extracellular matrix FGF: Fibroblast growth factor FGF-1: Fibroblast growth factor-1 FGF-2: Fibroblast growth factor-2 HGF: Hepatocyte growth factor HIF: Hypoxia-inducible factor
MCP-1: Monocyte chemoattractant protein-1 MSC: Mesenchymal stromal cell
PDGF: Platelet derived growth factor PDGF-B: Platelet derived growth factor-B PDGF-C: Platelet derived growth factor-C TGF-β: Transforming growth factor-β TGF-β1: Transforming growth factor-β1 TGF-β2: Transforming growth factor-β2 VE-cadherin: Vascular endothelial-cadherin VEGF: Vascular endothelial growth factor
VEGF-R1: Vascular endothelial growth factor-receptor 1 VEGF-R2: Vascular endothelial growth factor-receptor 2 VSMC: Vascular smooth muscle cell
ii INDEX
1. INTRODUCTION ... 1
1.1 Neovascularization ... 1
1.1.1 General Aspects ... 1
1.1.2 The Angiogenic Process ... 2
1.1.3 The Arteriogenic Process ... 3
1.1.4 Angiogenesis, Arteriogenesis and Ischemia ... 4
1.1.5 Proangiogenic Factors ... 5
1.2. Therapeutic Angiogenesis ... 7
1.2.1 UCX® ... 8
1 1. INTRODUCTION
1.1 NEOVASCULARIZATION
In a context of ischemia, one of the main therapeutic goals is to promote revascularization to ensure appropriate oxygenation to the affected tissues. This can be achieved through three mechanisms: vasculogenesis, angiogenesis and arteriogenesis1–3. These represent different features of an integrated process2 and constitute the physiological response to ischemia4,5; however, this response may not be sufficient to compensate the lowering of blood flow6.
1.1.1 GENERAL ASPECTS
During the pre-natal phase, the vascular network begins its formation through vasculogenesis, which includes migration and in situ differentiation of mesoderm-derived precursor cells (angioblasts) into endothelial cells (ECs)1,7,8 that assemble in a primitive vascular labyrinth1,2,7,8. The subsequent growth and expansion of new capillaries from pre-existent ones is termed angiogenesis2,3,7,9–12.
The third process, here referred, that contributes to form a mature vascular network, is arteriogenesis, in which occurs the recruitment of pericytes and vascular smooth muscle cells (VSMCs)3,10,12. These cells cover nascent endothelial cell channels3,10, allowing formation of larger collaterals between existing arterial networks12, providing stability3. In adults, vessels are quiescent; however neovascularization can occur also through these three processes, angiogenesis being the most important1–3. Post-natal vasculogenesis involves recruitment of bone marrow-derived endothelial progenitor cells2,10. Physiological angiogenesis in adults occurs, for example, during wound repair and women’s menstrual cycle13.
Angiogenesis is a complex/multi-step process, regulated by a dynamic balance between proangiogenic (like Vascular Endothelial Growth factor – VEGF and Hepatocyte Growth factor - HGF) and antiangiogenic factors (like angiosatin and endostatin)1,2,13–15, interacting with multiple cells7. In fact, ECs alone can initiate but not complete angiogenesis1, as peri-ECs and matrix components have essential roles7.
Once this balance is disrupted, pathological angiogenesis occurs14,15: cancer is an example in case of excessive angiogenesis and on the opposite side, peripheral arterial disease2,15.
2 The modulation of angiogenesis, whether by promoting therapeutic angiogenesis or by preventing pathologic angiogenesis has been extensively studied14.
1.1.2 THE ANGIOGENIC PROCESS
Once a vessel senses a proangiogenic stimulus, like VEGF, Angiopoietin-2 (ANG-2), Fibroblast Growth Factors (FGFs) or chemokines, released, for example, by a hypoxic signal10, vasodilation and an increase in vascular permeability occur in response to nitric oxide1,7and VEGF, respectively1,7,10.
This increase in permeability leads to redistribution of intercellular adhesion molecules, such as platelet endothelial cell adhesion molecule -1 (PECAM-1) and vascular endothelial (VE)-cadherin7, causing loss of cell-cell junction contacts between ECs and extravasation of plasma proteins1,10,11,15.
These proteins consist in several proteases (such as metalloproteinases –MMPs)1,3,7,15 that mediate the degradation of the basement membrane and surrounding extracellular matrix (ECM)7,11.
With this step occurs detachment of mural cells3,7, stimulated by ANG-2 and released by ECs3.
The degradation of the basement membrane allows the migration of ECs3,7 and the activation or release of proangiogenic molecules sequestered in the ECM, such as VEGF and FGFs3,10. On the other hand, antiangiogenic molecules such as protease inhibitors (like tissue-localized inhibitors of metalloproteinases - TIMPs) are also required to prevent excessive proteolysis7 and inappropriate sprouting3. During this process, some ECs become selected as the tip cells, which protrude filopodia, guiding sprouts properly3,10,15. The ECs surrounding the tip cells assume subsidiary positions as stalk cells; these trail tip cells and are more proliferative, thus expanding the growing endothelial channel and establishing the lumen3,10,11,15.
Tip and stalk cells are transient phenotypes3,11; in fact not all ECs exposed to proangiogenic signals become sprouting tip cells, thus a tight regulation is required, primarily through VEGF/VEGF-R2 (Vascular Endothelial Growth Factor-Receptor 2)3,11 and Notch pathways3,15. Tip cells contact other tip cells to add new vessel circuits to the existing network and once this contact is established, VE-cadherin containing junctions consolidate the connection3.
3 The ultimate stage in the angiogenic process is the maturation which includes deposition of ECM and basement membrane, recruitment of mural cells, reduced EC proliferation (resuming ECs to a quiescent state) and increased formation and reestablishment of cell junctions3,10.
Pericytes establish direct cell-cell contact with ECs in capillaries, sharing a common basement membrane, and their recruitment is dependent on platelet-derived growth factor B (PDGF-B), transforming growth factor β (TGF-β)3,7,10,16, ANG-1, ephrin-B2 and Notch10, while VSMCs cover arteries and veins and are separated from ECs by a matrix3. These mural cells stabilize vessels and can suppress endothelial proliferation7,10.
Maturation allows the remodelling into a hierarchically branched network and the adaptation of the vascular pattern to the local tissue needs3. This remodelling is also influenced by the hemodynamic forces intrinsic to blood flow3,15.
In fact, the last step is the onset of blood flow3. Since ECs are equipped with oxygen sensors (e.g: prolyl hydroxylase domain 2 - PHD2) and hypoxia-inducible factors (e.g: hypoxia-inducible factor-2α - HIF-2α), ECs can re-adjust their shape to optimize blood flow, or vessels may regress if they are unable to become perfused10. With perfusion, oxygen and nutrient delivery, VEGF expression is decreased and the endothelial oxygen sensors inactivated3. Without further proangiogenic stimuli15, there is a shifting EC behaviour toward a quiescent phenotype3.
Quiescent ECs have long half-lives7,10,17,18 and their survival is based on the autocrine action of maintenance signals such as FGFs, Notch, VEGF, ANG-1; these latter two are also released by pericytes10.
1.1.3 THE ARTERIOGENIC PROCESS
After narrowing or occlusion of an artery, there is an increase in fluid shear stress against the vessel wall2,19 that triggers arteriogenesis through activation of ECs2,19–21.
Activated ECs express chemokines (monocyte chemoattractant protein-1, MCP-1, being of major importance) that generate a chemotactic gradient for the recruitment of monocytes, and other leukocytes may also be involved2,7,19–22. Adhesion molecules, such as Intercellular Adhesion Mollecule-1 (ICAM-1)2,7,23, are also upregulated. Taken together these stimuli lead to adhesion, infiltration and accumulation of monocytes in the perivascular area2,7,19,20,23,24. Leukocytes produce multiple cytokines that induce proliferation of ECs and upregulate FGF-2, PDGF-B and TGF-β1, which stimulate
4 VSMC growth7. Simultaneously, monocytes also release proteases2,20,21, that degrade extracellular structures, creating space for the artery to expand7,19. Then, the maturation phase is characterized by i) rearrangement of VSMCs in circular layers; ii) establishment of contacts cell-to-cell and iii) synthesis of elastin and collagen to create the new scaffold for the much larger vessels24. Finally, the last phase of remodelling is characterized by the pruning of vessels that had initially participated in the remodelling but that are eliminated by the competition for flow24.
This cascade of events leads to an increased vessel lumen and wall thickness20, restoring blood flow (about four to six weeks after the occlusion setting of a large artery in animal models)19.
1.1.4 ANGIOGENESIS, ARTERIOGENESIS AND ISCHEMIA
Once an ischemic event happens, low levels of oxygen are prevalent in the tissues and hypoxia plays an important role in regulating proangiogenic factors, like VEGF5,7,15,20,25: accumulation of VEGF mRNA occurs both in vivo and in vitro during hypoxia7,20. Several other genes, directly or indirectly involved in angiogenesis, are also upregulated in response to hypoxia. These include VEGF-R1, VEGF-R2, ANG-27,16,25 and HIF-15. In fact, HIFs orchestrate adaptive responses to changes in oxygen tension by regulating the expression of genes involved in cell survival, metabolism, and angiogenesis3,20. Although ischemia is often present when collaterals develop24, arteriogenesis is not considered hypoxia-driven like angiogenesis2,26. In fact, fluid shear stress constitutes the main arteriogenic stimulus19,24,27, although arteriogenesis may also be triggered by circulating factors produced in distant ischemic territories2.
Through arteriogenesis, a bypass is developed around the occlusion site2,20 and pre-existent collateral vessels can enlarge up to 25 times their original size21, resulting in high-conductance vessels that rapidly increase blood flow, unlike capillaries formed via angiogenesis21. Furthermore, the time course of angiogenesis and arteriogenesis is different2: in a rabbit model, for example, capillaries were formed five days after femoral artery removal, while collaterals developed after ten days9.
5 1.1.5 PROANGIOGENIC FACTORS
VEGF, ANG, TGF-β, PDGF, FGF and HGF are important players in the angiogenic process and we will, very briefly, describe them in this subchapter.
1.1.5.1 VEGF
VEGF, also known as VEGF-A is the main component of the VEGF family and one of the most well-known promoters of angiogenesis in health and disease10,11. VEGF alone can initiate although not complete angiogenesis1.
VEGF has two main receptors: VEGF-R2 (also known as fetal liver kinase-1 - Flk-1, or kinase insert domain receptor - KDR) and VEGF-R1 (also known as fms related tyrosine kinase-1 – Flt-1)2,10,11,16.
VEGF-R1 and VEGF-R2 are expressed in ECs, in early precursors of the closely linked hematopoietic lineage2,13 and in monocytes2,16.
Among proangiogenic factors, the VEGF-related factors are distinguished by their specificity for ECs13.
VEGF-R2 has a lower affinity for VEGF than VEGF-R1, but it has a stronger tyrosine kinase activity10,11. Therefore VEGF functions are mainly attributed to its binding to VEGF-R2 and these include regulation of survival and proliferation of ECs11,15,16,25. On the other hand, VEGF-R1 shows a higher affinity for VEGF and a lower kinase activity and it may act as a decoy receptor, moderating the amount of free VEGF available to activate VEGF-R210,11,15,16.
1.1.5.2 ANG
The human ANG family includes Tie receptors and ANG ligands10,16. ANG-1 is a Tie2 agonist and ANG-2 functions as a competitive ANG-1 antagonist in a context-dependent manner10.
ANG-1 participates in the maturation and stabilization of the vascular networks, playing a role in angiogenesis later than VEGF 1,13.
In the presence of an angiogenic stimuli, ANG-2 is released from tip cells1, possibly blocking a constitutive stabilizing or maturing function of ANG-1, thus allowing vessels to remain in a more plastic state. Here they may be more responsive to an angiogenic signal provided, for example, by VEGF13, promoting therefore mural cell detachment, vascular permeability and EC sprouting10.
6 1.1.5.3 TGF-β
Members of the TGF-β family are involved in a lot of processes regarding cell proliferation, differentiation and regulation28,29. Their pro- and anti-angiogenic effects are context-dependent10,29.
TGF-β2 has been shown to modulate EC behaviour and to induce VEGF secretion30.
1.1.5.4 PDGF
PDGFs and their receptors are involved in multiple cellular processes during development and in pathological mechanisms31.
PDGF-B, released by activated ECs, serves as a chemoattrant for mural cells that express PDGF-Rβ, contributing to vessel maturation1,7,10,15,18,32 and can induce the secretion of VEGF from these same cells32.
The role of PDGF-C has been more investigated in pathological angiogenesis than in developmental angiogenesis, especially as a mediator of angiogenesis independent from VEGF in cancer33.
PDGF-C exerts its angiogenic activity through multiple actions in different target cells, like recruitment of endothelial progenitor cells, proliferation and migration of ECs and pericytes33.
1.1.5.5 FGF
FGFs and their receptors control a wide range of biological functions10. Particularly, FGF1 and FGF-2 have shown to promote angiogenesis in animal models of ischemia17. FGF-2 was among the first discovered angiogenic factors10,17 and has angiogenic properties10 directly through stimulation of migration and proliferation of ECs34 and indirectly through induction of angiogenic factors from other cell types10.
Moreover, FGF-2 shows arteriogenic properties10 through promotion of proliferation of VSMCs and fibroblasts, that participate in arteriogenesis34.
1.1.5.6 HGF
HGF is a pleiotropic factor that acts on a wide variety of cell types14 and regulates many processes, such as cell growth and motility35.
HGF stimulates EC migration, proliferation, and tube formation in vitro36.
The angiogenic activity of HGF is a combination of direct actions on ECs37 by binding to its receptor (c-met)14 and indirect effects, including induction of VEGF from VSMCs14,37.
7 In vitro, c-met showed downregulation in quiescent ECs, but upregulation in activated ECs14, and in vivo, upregulation in a rat model of myocardial ischemia38.
Interestingly, HGF showed a more potent proliferative activity on ECs than that of VEGF in human aortic ECs in vitro and in a rabbit model of hindlimb ischemia in vivo35.
1.2 THERAPEUTIC ANGIOGENESIS
Concerning limb ischemia, many patients suffering from peripheral arterial disease are not suitable candidates for either surgical or endovascular treatment, requiring amputation as their only option39,40. In this context, therapeutic angiogenesis surfaces as a novel and promising strategy40.
Therapeutic angiogenesis aims to induce the formation of new vessels in ischemic tissues21,41 through administration of proangiogenic factor in the form of recombinant proteins26,42,43 or by gene therapy44,45 or by cell therapy46–51, altering the local milieu to provide a proangiogenic environment21. Not only therapeutic angiogenesis aims to promote angiogenesis in ischemia, but also vasculogenesis and arteriogenesis, as they are part of an integrated process to ensure neovascularization2,52.
Recombinant proteins have shown some disadvantages due to their limited tissue half-life, which may require repeated administrations2,53.
Gene therapy, through viral or non-viral vectors, can improve the duration of transgene expression, facilitating sustained and local production of these angiogenic factors in comparison to direct protein administration2,12.
Cell therapy has been performed through introduction of cells with a stem potential from different origins, mainly from bone marrow or peripheral blood39,54–56.
Secondary effects of therapeutic angiogenesis have been reported2,39,50,57, like aggravation of restenosis in peripheral arterial disease or worsening of atherosclerosis2 or local temporary side effects, such as edema45. Besides, too high expression of an angiogenic factor may have the opposite desired effect as seen with a viral vector overexpressing VEGF that resulted in accelerated limb loss after administration in mice2.
The first study on therapeutic angiogenesis in humans was performed by Isner et al in 1996 using a VEGF plasmid58. Since then, multiple clinical trials have been performed with gene therapy using, for example, HGF59 and FGF-260,61, although without showing sustained success.
8 Regarding cell therapy in humans, the first large study was TACT (Therapeutic Angiogenesis Using Cell Transplantation) in 200262, using autologous bone marrow derived cells, and it showed a promising outcome12,63. Results from the clinical trials have been promising51, although still far from what was expected, and multiple variables have been suggested to explain the negative outcomes, such as the use of only a single factor5,64, dose administered2,65,66, duration of expression, mode of delivery, lack of objective parameters2,49, different trial end points and patient heterogeneity2.
Although bone marrow derived cells are the most commonly used in multiple trials21, as they are thought to be a supply of endothelial progenitor cells and multiple angiogenic factors62, other sources have surfaced as being more advantageous, like umbilical cord65,67–69. This source presents wider availability, no surgery risk, higher concentration of stem cells and greater proliferative capacity and immune tolerance67,70.
1.2.1 UCX®
ECBio, a biotechnology company, has developed proprietary technology to consistently isolate, expand, and cryopreserve a well-characterized population of human stem cells derived from the umbilical cord (Wharton’s jelly), named as UCX® cells71.
The technology used for UCX® isolation is different from other umbilical cord derived Mesenchymal Stromal Cells (MSCs) isolation methods. Very briefly: i) the introduction of a three-stage strategy for cell recovery was introduced in order to make the method 100% reliable; ii) the isolation begins with peeling off the amniotic membrane which reduces the frequency of microbial contamination and augments the purity of the resulting UCX® population by eliminating epithelial progenitors; iii) tissue incisions and/or tissue mincing/ crushing steps are avoided since they could hinder its application in good manufacturing practice settings and jeopardize stem cell phenotype due to excessive mechanical manipulation; and iv) the use of an optimized ratio between tissue mass, digestion enzyme activity units, overall solution volume and void volume allowing UCX® cell-specific release and intact cord vessels that will reduce contamination with endothelial and sub-endothelial cells from umbilical arteries and vein71.
UCX® cells are a homogenous population of stem cells that complies with the current definition of MSCs as established by the International Society for Cellular Therapy (ISCT). Namely cells that adhere to a plastic surface; at least 95% of UCX® cells in the population are positive for the cell surface markers CD44, CD73, CD90 and CD105 and
9 less than 2% positive for CD14, CD19, CD31, CD34, CD45 and HLA-DR; and are capable of undergoing tri-lineage differentiation into adipocytes, chondrocytes and osteoblasts71.
Importantly, it was demonstrated that UCX® may be a new approach for treating both local and systemic manifestations of inflammatory arthritis71. It was also shown that UCX® cells are immunosuppressive and have low immunogenicity, relevant for allogeneic applications71.
Furthermore, by using a murine model of myocardial infarction, Nascimento et al showed a cardioprotective effect of UCX® that is exerted through paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis in the heart, increase proliferation and activate a cardiomyogenic gene-expression program of myocardial resident cardiac progenitor cells72. In this particular work it was shown that UCX® increases capillary density in the infarcted myocardium and UCX® cells express high levels of angiogenesis-associated transcripts such as VEGF, ANG, HGF, c-met, FGF-2 and TGF-β72.
According to those results, our main goal is to determine whether UCX® has clinical use in the treatment of lower limb vascular insufficiency.
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