Bio of Cancer Exam 4 Material

Activating Invasion and Metastasis

"Sooner or later during the development of most types of human cancer, primary tumor masses spawn pioneer cells that move out, invade adjacent tissues, and thence travel to distant sites where they may succeed in founding new colonies." "The capability for invasion and metastasis enables cancer cells to escape the primary tumor mass and colonize new terrain in the body where, at least initially, nutrients and space are not limiting." Hanahan and Weinberg, 2000 • "may succeed": actually it is a really hard process, some cancer cells won't make it

Inducing Angiogenesis

"The oxygen and nutrients supplied by the vasculature are crucial for cell function and survival...during organogenesis, this closeness is ensured by coordinated growth of vessels...once a tissue is formed, the growth of new blood vessels - the process of angiogenesis - is transitory and carefully regulated." "In contrast, during tumor progression, an ''angiogenic switch'' is almost always activated and remains on, causing normally quiescent vasculature to continually sprout new vessels that help sustain expanding neoplastic growths." Hanahan and Weinberg, 2000 and 2011

Steps involved in the tumor invasion - metastasis cascade

1. Localized invasion 2. Intravasation 3. Transport 4. Extravasation 5. Formation of micrometastasis 6. Colonization

Triggers of Angiogenesis in Cancer

1. Loss of Tumor Suppressors and Activation of Oncogenes a. Aberrant Production of Growth Factors (oncogenic proteins) b. Loss of Tumor Suppressor Proteins 2. Hypoxia

Overview of Angiogenesis

1. Tumor cells release pro-angiogenic factors, such as VEGF, which diffuse into nearby tissues and bind to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. 2. Such interactions between endothelial cells and tumor cells lead to the secretion and activation of various proteolytic enzymes, such as MMPs, which degrade the basement membrane and the extracellular matrix. 3. Degradation of the basement membrane allows activated endothelial cells, which are stimulated to proliferate by growth factors, to migrate towards the tumor. 4. Integrin molecules help to pull the sprouting new blood vessel forward. 5. The endothelial cells deposit a new basement membrane and secrete growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel

Analogy for Angiogenesis

Airplane • the airplane keeps going and going and runs out of fuel • angiogenesis is like the extra fuel tank, so it never runs out House • building a house in the middle of no where with no pipes or electrical lines • in order for that house to survive they need to bring in those pipes and wires and add a road • this is angiogenesis

4 Steps of Angiogenesis

The process of angiogenesis primarily consists of four distinct sequential steps: 1. Endothelial cell activation. 2. Degradation of basement membrane glycoproteins and other components of the extracellular matrix surrounding the blood vessels by proteolytic enzymes. 3. Endothelial cell migration and proliferation. 4. Endothelial cells tube formation.

Angiogenesis and Cancer

• Cancers activate a developmental program known as the angiogenic switch to continuously grow and expand a vasculature network was the tumor grows. • Activation of the angiogenic switch is complex and involves increased production of proteins that stimulate vascular growth, and decreased expression of inhibitory proteins that discourage vascular expansion.

Blood Vessel Structure

• endothelial cell (EC) • basement membrane • adhesion protein molecules

Analogy for Resisting Cell Death

• wear and tear is building up and at the same time its never getting to the point where the car dies • immortality • brakes don't work (loss of tumor suppressor genes), accelerator is pushed down (oncogenes), tank is never empty (angiogenesis)

Improved Nutrient Supply

○ A tumor's need for nutrients grows in proportion to its volume, but its ability to absorb diffusing substances from the surrounding tissue is proportional to its surface area. • This imposes a maximum size to which the tumor can grow before it experiences nutrient deficiency: > Some of the tumor cells (usually those towards the center of the tumor, where nutrient levels are at their lowest) will not have sufficient nutrients to continue to proliferate and will be quiescent (inactive). > If the nutrient supply is not improved, necrosis (cell death caused by insufficient nutrition or injury) will set in, leading to the development of a necrotic core of dead cells. ○ In order for a tumor to survive, grow, and metastasize it must induce the growth of new blood vessels. • This allows the tumor to progress from the avascular (lacking blood vessels) to the vascular (possessing a blood supply) state

a. Aberrant Production of Growth Factors (oncogenic proteins)

○ Aberrant production of growth factors, in addition to acting in an autocrine manner to stimulate proliferation of tumor cells, can also act in a paracrine manner to stimulate the growth of endothelial cells. • Oncogenic proteins including receptor tyrosine kinases (ex. EGF-R), intracellular tyrosine kinases (ex. Src), intracellular signal transducers (ex. Ras), and transcription factors (ex. Myc, Fos, and Jun), have been shown to up-regulate the key angiogenic inducer, VEGF. >Myc via Ras activation, also inhibits Tsp-1.

1. Loss of Tumor Suppressors and Activation of Oncogenes

○ Activation of various oncogenes and loss of tumor suppressors contribute to the modification of the angiogenic switch and tip the balance towards angiogenesis. • Unlike the well known direct contribution of oncogenes and tumor suppressors to proliferation, apoptosis, and differentiation, the roles of these genes in angiogenesis are still being elucidated. • Many of the oncogenes and tumor suppressor genes previously discussed (and in the context of the other hallmarks of cancer) have been shown to also be involved in angiogenesis.

Inducing Angiogenesis: Definition

○ Although diffusion can accommodate the needs of small tumors (<2 mm in diameter) to import (oxygen and nutrients) and export (carbon dioxide and metabolic wastes), growth beyond the diameter of a millimeter or so requires the acquisition of a network of blood vessels, a vasculature. • diffusion doesn't work on bigger tumors b/c the inner cells are getting further away from the healthy cells so they cannot get nutrients (diffusion doesn't make it to inner tumor) • this would normally result in death, this is why angiogenesis comes into play

Vasculature in Cancerous Tissue (Angiogenesis)

○ Angiogenesis and tumor progression are very closely linked with each other. • Tumors cannot grow beyond approx. 2 mm in diameter or metastasize (spread) without a blood supply (Folkman, 1971). • Cancer cells have overcome this problem by acquiring the ability to activate angiogenesis. • The blood supply will provide the cancer cells the oxygen and nutrients needed for survival, as well as a means for the primary tumor cells to utilize the vasculature as a transport system to setup up secondary tumors at other sites throughout the body.

Angiogenesis Mechanism

○ Angiogenesis is a very complex process and involves interactions and heterotypic signaling between various biological components, such as: > several cell types >soluble angiogenic factors >extracellular matrix components ○ In normal conditions, angiogenesis only occurs during embryonic development, the female reproductive cycle and wound repair. ○ However, aberrant angiogenesis is a key mediator and a major process in cancer development.

Angiogenesis

○ Angiogenesis is the process by which new blood vessels are formed via the invasion of endothelial cells from preexisting blood vessels in response to extracellular signals • (Greek; angio = vessel, genesis = creation). • In comparison, neovascularization is the initial formation of new blood vessels that mainly occurs during embryogenesis. • In the adult, the angiogenesis process takes place during growth and development such as with the female menstrual cycle and wound healing. • Additionally angiogenesis occurs in pathological disease conditions such as cancer, macular degeneration, psoriasis, and rheumatoid arthritis.

3. Transport

○ Cancer cells travel through the blood or lymph until they anchor to a solid supporting tissue. • Although both blood and lymph are responsible for transport, most of the distant metastases are caused by circulation through the bloodstream. • At this stage, most of the cancer cells can be lost or destroyed due to hostile conditions. • However, surviving cancer cells get lodged in the first set of capillaries they encounter (mainly due the large cells blocking the small passage of the capillaries) and form microthrombi.

adhesion protein molecules

○ Cell to cell contacts and cell to basement membrane contacts, mediated by adhesion protein molecules (such as cadherins and integrins), are extremely important. • Loss of either or both can lead to local destabilization of the endothelium and EC apoptosis. ○ The support cells play a particularly important role in maintaining blood vessels in the stable state, and may be involved in the regulation of blood flow.

b. Loss of Tumor Suppressor Proteins

○ Certain tumor suppressor proteins normally up-regulate/increase angiogenic inhibitors, but when these are mutated, anti-angiogenic activity decreases. • For example, p53, normally binds to and activates the promoter of the TSP-1 gene. • Mutations in the p53 gene, commonly associated with the cancer phenotype, result in a decrease of the angiogenic inhibitor so that the angiogenic switch favors angiogenesis. ○ The TME conditions may also play a role in triggering angiogenesis (i.e. hypoxia

Angiogenesis: Quiescence

○ Finally, the endothelial cells in the new vessels revert to a quiescent phenotype.

Malignant Cells

○ Fully malignant cells, besides having acquired all the properties previously mentioned, are invasive and can be metastatic; the final of the six hallmarks of cancer. • Malignant cell invasion is the active process by which such cells translocate into and through extracellular matrix barriers. • Metastasis is the process by which malignant cells depart from the primary tumor and travel to distant sites within the body to form secondary tumors. ○ Like the formation of the primary tumor mass, successful invasion and metastasis depend upon all of the other five acquired hallmark capabilities. • "cancer" is ultimately it obtaining this last, spreading hallmark (otherwise its just a benign tumor) • although hallmarks do occur in non-specific order, this is usually last or 2nd to last step

LEGO Analogy

○ Imagine a pile of Lego bricks: • Some pieces are big, some are small; some are long, some are wide; some are traditional bricks, some are specialized. • A nearby box displays a completed Lego spaceship, but no instructions on how to build it. > A reductionist can count, measure and document the bricks. They can describe the bricks with absolute accuracy. > But ask the reductionist how these bricks combine to build our spaceship, and it becomes less clear; some bricks look like they might form a wing, maybe others look like the cockpit, but ultimately the reductionist's list of bricks alone does not contain enough information to build the spaceship.

Factors that Stimulate Cell Motility

○ In addition to an ability to degrade the extracellular matrix, malignant cells must have the ability to migrate through the matrix. ○ There are several factors that contribute to this migration by stimulation of cell motility. • These factors, such as HGF/SF (hepatocyte growth factor/scatter factor) and IGF-1 (insulin-like growth factor type 1), can be secreted by the tumor cells and function in an autocrine manner. • Similar in idea to that of GFs produce by cancer cells; the cancer cell produces a molecule that then acts upon itself.

Tissue Inhibitors of Metalloproteinases (TIMPs)

○ In normal cells, the expression of tissue inhibitors of metalloproteinases (TIMPs), inhibit MMPs by direct binding resulting in inactivation. • A tip in the balance between MMPs and TIMPs can signal invasion.

Cancer - A Heterotypic Approach

○ In normal tissues, several types of cells interact/communicate with one another (termed heterotypic signaling) in a controlled and regulated manner to provide normal structure and function. • The same idea holds true for cancer cells and tumors: > It is increasingly apparent that tumors are complex ecologies of different cell types (both normal and cancerous) and that tumor growth and malignancy requires an appropriate support structure, the tumor microenvironment (TME). • The tumor microenvironment consists of microvasculature cells, fibroblasts, other stromal elements and pro-inflammatory leukocytes

1. Localized Invasion

○ In this process, the small tumor breaks through the basement membrane barrier.

Inflammatory Cells

○ Inflammatory cells, such as macrophages, are able to produce both anti- and pro-angiogenic molecules. Pro-Angiogenic • Can produce molecules that alter the extracellular matrix, proteases (induce migration of endothelial cells) and growth factors (induce mitosis and differentiation of endothelial cells). Anti-Angiogenic • Can produce both thrombospondin-1 (Tsp-1) and vascular endothelial growth factor (VEGF)

Role of Integrins

○ Integrins are transmembrane receptors that bind to extracellular matrix components such as laminin, fibronectin, and collagens. • The binding of integrins to these matrix components activates signal transduction pathways, which activate genes involved in cell cytoskeletal organization and support. • Integrins consist of various subunits, and different combinations of these subunits result in different integrin subtypes. • Each integrin subtype has a distinct substrate preference and thus, different integrin combinations are expressed in normal cells, while other combinations are expressed only in malignant cells. ○ Altered integrin receptor expression in tumor cells can enable the mobility and invasion of metastasizing cells by modifying membrane distribution and/or allowing adherence to different ECMs. • Regulation by the cancer cell must result in precise intermediate strengths of adhesion to produce the maximum rate of cell migration, allowing cells to advance their leading edge and to release their lagging edge.

Where & How of Metastasis

○ Normally, metastasis occurs via the blood circulation, but it can also occur through the lymphatic system. ○ Interestingly, not all the cells within a primary tumor can metastasize. • not all will survive the rigorous process • cells are at the mercy of the blood/lymph flow • not a large mass, every cell for themselves ○ This is consistent with the multi-step process of carcinogenesis, which suggests that additional genetic alterations are required for tumor metastasis.

Angiogenesis ♡ Metastasis

○ Not only does angiogenesis provide the tumor cells with an almost unlimited supply of oxygen and nutrients for growth and survival but also aids in the process of metastasis (Chapter 14). ○ The proximity of large numbers of blood vessels increases the likelihood of tumor cells entering the bloodstream and being transported to other parts of the body. • Allows for the tumor cells to establish secondary tumors. • The more malignant the tumor is, the greater its angiogenic potential is. ○ The ability to induce angiogenesis occurs as a mid-stage event in tumor development, prior to tissue invasion. ○ Angiogenesis is an essential component of the metastatic pathway (Chapter 14). • The newly formed blood vessels allow for tumor cell invasion and metastasis by providing the principal route by which the cells exit the primary tumor site and enter the circulation.

Lego Spaceship = Tumor

○ Now imagine our spaceship is a tumor. • A single human cell contains over 20,000 different protein-coding genes, over 100,000 protein isoforms, and around 1 x 10^10individual protein molecules. • A small (10 mm3) tumor is composed of approximately 1 x 10^9cells; that means a single tumor contains around 1 x 10^19 protein molecules. • If a small tumor were built from Lego bricks instead of proteins, it would fill 15 Grand Canyons; most clinical tumors would be the size of a small country. >At this scale, the emergent tumor cannot be captured by a reductionist list - no matter how precise.

Angiogenesis: Proliferation & Recruitment

○ Once a vessel is stimulated, endothelial cells begin to proliferate, elongating the developing, tube-like structures (caused by integrin proteins). • These new tubes connect into loops that allow blood flow. ○ The recruitment of smooth muscle cells and other cell types (by growth factors such as PDGF) completes the angiogenesis process by forming the new basement membrane, resulting in a mature blood vessel.

basement membrane

○ Outside the endothelium is an extracellular lining called the basement membrane. • Separates the EC from the surrounding connective tissue. • Composed of protein fibers, mainly laminin (smaller and Y-shaped) and collagen (long fibers), and may also contain support cells. • The basement membrane serves as a scaffold on which the EC rest and helps to maintain the endothelium in its functional state.

Invasion & Metastasis

○ Primary tumors not only disseminate cancer cells to the immediate local environment but distribute them through the blood and lymph to distant organs. • Once lodged in the distant environment, some of this emigrant cancer cells surmount the challenges of adaptation to the new environment and proliferate, establishing metastatic foci of the primary tumor. • last hallmark but sorta 2 different ones 1. invasion: cells translocate into and through extracellular matrix barriers. 2. metastasis: departure from primary tumor and travel to make secondary tumors

Factors That Have Angiogenic Activity

○ Several factors that have angiogenic activity have been identified. ○ The first to be identified were the fibroblast growth factors (FGFs). ○ The most important (and specific to endothelial cells) one discovered is VEGF.

Metastases

○ The distant settlements of tumor cells, termed metastases, are the cause of 90% of human cancer deaths.

Local (Tissue) Invasion

○ The first steps involved in malignant cell tissue invasion are the acquired abilities to attach to the extracellular matrix components and disrupt the basement membrane. • Both involve changes in the expression of integrins and cellular adhesion molecules (CAMs). • CAMs are involved in cell to cell adhesion interactions and produce anti-metastatic signals through these interactions. >In metastasis, the loss of CAM functions causes a decrease in cellular adhesiveness. • Integrins are involved in extracellar matrix binding

fibroblast growth factors (FGFs)

○ The first to be identified were the fibroblast growth factors (FGFs). • Stimulate endothelial cell migration by inducing the production of proteases by endothelial cells and stimulate tube formation.

Tumor Microenvironment and Metastasis

○ The interaction of tumor cells with the tumor microenvironment (TME) affects the metastatic ability of individual cancer cells. • the TME has helped the tumor to survive and grow, but not it has become an obstacle course ○ Although the genetic alterations involved in tumor initiation and formation vary between different cancer types, the alterations required for tumor invasion and metastasis are similar. • will have to re-colonize and build TME all over again

Role of CAMs

○ The main role of the CAMs is to tether cells to surrounding tissue. • The most common CAM implicated in metastasis is E-cadherin, which is found in all epithelial cells. • In normal cells, E-cadherin acts as a bridge between adjacent cells, enabling cytoplasmic contact and sharing intracellular signaling factors responsible for inhibiting invasion and metastatic capability. • Acts as a tumor suppressor protein. • Most epithelial cancers show a loss of E-cadherin function and this elimination plays a significant role in metastatic capability.

endothelial cell (EC)

○ The most essential component of blood vessels is the endothelial cell (EC). ○ Every vessel, from the aorta down to the smallest capillaries, consists of a monolayer of EC (called the endothelium), arranged in a mosaic pattern around a central lumen, through which blood can flow. • In the smallest vessels, a cross-section of the endothelium may consist of a single EC, which has wrapped around to form a lumen. >The endothelium controls the passage of nutrients, waste, white blood cells, and other materials between the bloodstream and the tissues.

Cancer - Reductionist Approach

○ The traditional approach to understanding the cancer process was from a reductionist point of view. • By reducing an object to its constituent parts, we can attempt to understand the whole. • This approach has obviously led to our knowledge of oncogenes and tumor suppressor genes and their association with the various hallmarks of cancer already discussed. • But, it does not provide us with a complete understanding of cancer process and how everything comes together to work in coordination with one another.

2. Intravasation

○ The tumor cells move through the walls of the capillaries or lymphatics into the circulatory system. • This is a critical step in this pathway and it involves a complex, morphological change, wherein the cancer cell acquires properties of invasiveness and cell motility. • This enables the cancer cell to push its way through the capillary wall and into the circulatory system.

TME Role in Angiogenesis

○ The tumor microenvironment (TME) is created by a complex and dynamic network of inflammatory cells, growth factors and matrix remodeling enzymes. • Inflammatory cells, such as macrophages, are able to produce both anti- and pro-angiogenic molecules.

The Tumor Microenvironment

○ The tumor microenvironment (TME) plays a key role in tumor survival and angiogenesis. •There are a large number of pro-angiogenic and anti-angiogenic factors: > some of which are produced by the tumor > some of which are produced by host cells in response to the tumor > some of which are present in normal tissue. • the cells of the tumor microenvironment have no ability to question the signals/instructions >they do what is considered their normal function, but at an inappropriate time/location/frequency

Normal Cell vs. Cancer Cell Protein Balance

○ There are several steps involved in tumor invasion and metastasis, each requiring the expression of different proteins involved in cell to cell adhesion, cell-matrix interaction, proteolysis, and motility. • In normal cells, there is a balance between the expression of these proteins. • In cancer, this balance is shifted; pro-invasive and pro-metastatic genes are up-regulated and anti-invasive and anti-metastatic genes are down-regulated.

4. Extravasation

○ This final step of migration is essentially similar to intravasation, in which the tumor cells eventually move into the tissues they are lodged in, typically lungs, brain, or liver. • The main difference is that the direction of movement is reversed. • Cancer cells in the microthrombi now push through the capillary wall and into the tissue microenvironment.

6. Colonization

○ This is the most complex and challenging stage mainly because the new environment may not always provide the necessary survival and proliferation factors needed for growth. • Most cancer cells usually die or survive for long periods as micrometastases (which are also much harder to detect).

Vasculature in Normal Tissue

○ To function properly, normal cells need sufficient oxygen and nutrients and waste removal, which are provided by the vasculature. • A normal cell must be located within approx. 200 μm (0.2 mm) of a capillary to survive in the body. • the max size of a tumor before needing angiogenesis is 2mm • 0.2 mm represents the distance that oxygen can effectively diffuse through living tissues. • Cells located further away than 0.2 mm from the vasculature will result in hypoxia, and potentially leading to necrosis or apoptosis. • Therefore these cell require a vasculature to survive.

Tsp-1 Gene Expression

○ Tsp-1 expression is been linked to many cancer genes (such as ras inhibition and p53 activation) and is down-regulated during tumorigenesis. • Hypermethylation of the TSP-1 gene and resultant decrease in expression has also been shown to play a role.

Tsp-1 Interactions

○ Tsp-1 interacts with a variety of molecules including: • structural components of the extracellular matrix • other extracellular proteins • cell receptors • growth factors • proteases. ○ These interactions can lead to a variety of outcomes such as: • Activation of receptors and downstream signaling pathways • sequestration and inactivation of growth factors and enzymes • alterations of protein localization • proteolytic processing and internalization

Tsp-1 Direct Function

○ Tsp-1 is an ECM protein associated with angiogenesis and wound healing/repair. • Inhibits migration, proliferation, apoptosis, and capillary tube formation of endothelial cells. > so inhibits division, death, movement, and apoptosis • Performs several functions in wound healing/repair; serves as a migratory matrix, enhancing neutrophil chemotaxis and inhibiting proteases.

Normal Angiogenesis: "angiogenic switch"

○ Under normal conditions, angiogenesis is suppressed by anti-angiogenic factors (ex. thrombospondin-1; Tsp-1). ○ When blood vessel formation is needed, there is a shift in the balance from the anti- to the pro-angiogenic factors (ex. vascular endothelial growth factor; VEGF). • Referred to as the "angiogenic switch." • This switch is a highly complex process, but not well understood. • Hypoxia (oxygen deficiency) in the tumor and surrounding inflammation is thought to be an important factor, stimulating production of pro-angiogenic molecules by the tumor cells

Matrix Metalloproteinases (MMPs)

○ Upon disruption of the basement membrane, malignant cells translocate across the extracellular matrix barriers by proteolysis and migration. • This translocation involves the expression of matrix metalloproteinases (MMPs). • Creates space for the advancing tumor cells. • These proteases are up-regulated in invasive tumors and allow the tumor cells to cleave and degrade the extracellular matrix. • Although some tumor cells can synthesize MMPs, more often tumor cells induce surrounding stromal cells to produce MMPs. • Several MMPs can be activated via RAS as well. ○ MMPs can not only degrade structural components of the ECM, but also cleave other proteins residing on the outside of cells (ex. endothelial cell growth factors), and thus are likely to play an important role in metastasis, as well as angiogenesis.

5. Formation of micrometastasis

○ Upon extravasation, the cancer cells are now able to reactivate the cell proliferation pathways and form a small tumor mass which either develops in the lumen of the capillary or through the vessel wall.

VEGF

○ VEGF is an endothelial cell mitogen and chemo-attractant • promotes: cell migration • inhibits: apoptosis • modulates: the permeability of the endothelial cell layer ○VEGFs bind to and activate their appropriate growth factor receptors (VEGF-Rs). • Basic transmembrane receptor tyrosine kinases that are able to form homodimers and heterodimers. >Dimerization of these receptors is accompanied by activation of receptor-kinase activity that leads to auto-phosphorylation of these receptors and activation of downstream transduction pathways. >Activation of VEGF-Rs results in expression of genes involved in cell migration, proliferation, survival, and mobilization of endothelial progenitor cells from the bone marrow into the peripheral circulation.

2. Hypoxia

○ When a condition such as hypoxia (lack of oxygen) is present, expression of p53 increases. ○ Recall that p53 will induce G1 arrest first, and ultimately apoptosis in hypoxic cells in the tumor's center. • Thus, in this region, strong selective pressure against functional p53 is created: a cell with newly mutated p53 can take advantage of this situation, continuing to proliferate in the face of arrested or dying competitors. • Hypoxia thus promotes the next step in tumorigenesis, selecting a cell that is more malignant by the loss of p53. >And normally, p53 activates the TSP-1 gene, which blocks angiogenesis. ○ In addition to p53 activation, hypoxia will cause the tumor cells to activate signal transduction pathways involved in the angiogenesis process. • In the case of hypoxia, the signal is mediated by the transcription factor hypoxia inducible factor-1 (HIF-1). >It has been shown that Ras can also activate HIF-1. • HIF-1 activates a number of hypoxia-response genes, including VEGF, by binding to specific sequences within the promoters of these genes, called hypoxia-response elements (HREs). >Thus hypoxia up-regulates the expression of angiogenic factors, like VEGF.

Angiogenesis: Initiation

○ When a tumor starts to produce angiogenic factors, it activates endothelial cells in the vasculature of the surrounding tissue to initiate angiogenesis. >alters the environment (TME) ○ This stimulus induces the endothelial cells of the "mother" vessels to change from a quiescent to an activated phenotype. ○ These endothelial cells produce proteolytic enzymes (called matrix metalloproteinases or MMPs), which break down the basement membrane and extracellular matrix. • Allows for endothelial cells to migrate into the surrounding tissue towards the growth stimulus. > Also promotes angiogenesis further by causing the release of various growth factors, including VEGF, PDGF, FGF, and EGF.