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Wound Healing is a complicated process which requires the coordination of several events including inflammation, new tissue generation, angiogenesis & matrix remodeling. Angiogenesis is crucial to the wound repair process; it is the growth of new blood vessels from the existing host vasculature. The main function of angiogenesis is to provide nutrition and oxygen to the growing tissue. It is a dynamic process regulated mainly by pro and anti-angiogenic proteins released by the cells in the serum and the surrounding extracellular matrix. Angiogenesis is known to be stimulated in the tissue repair process by a number of growth factors including vascular endothelial growth Factor (VEGF). Semaphorins can compete with VEGF to bond to Neuropilins and may inhibit angiogenesis or regulate vascular regression. Very few human studies have been undertaken to date in this field and most are from diabetic ulcers & not normal wound healing and most of the focus of ongoing studies is on the vascular initiation phase and not the remodeling and vascular regression phase. We test our hypothesis that Semaphorin 3B modulates vascular regression via binding to neuropilin-1 and Plexins on endothelial & vascular smooth muscle cells. We will characterize expression of Plexins, Semaphorin 3B & Neuropilin on endothelial cells and vascular smooth muscle cells using techniques such as Western Blotting Analysis which will be done on cell extracts from Human Endothelial cells & vascular smooth muscle cells. We will assess the functional effects of Semaphorin 3B on endothelial cells and Vascular smooth muscle cells. This project enables us to identify the expression of Sema 3B and its receptors on Endothelial cells and Vascular smooth muscle cells and to establish the functional effects of Sema 3B and thus aiding in our comprehension of the effects of Semaphorins and Plexins on Vascular Remodeling.
Wound Healing is a complicated process which requires the coordination of several events including inflammation, new tissue generation, angiogenesis & matrix remodeling . The classic model of wound healing is most commonly divided into three phases that are overlapping : The inflammatory phase, The Proliferative phase and finally wound remodeling .Â
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Kloth, L.C., McCulloch, J.M. (2002). Wound Healing, Alternatives in Management (3rd ed.).Â
Upon infliction of injury to the skin, complex biochemical events start to take place in a closely defined cascade to repair the damage . Within minutes after the injury, platelets aggregate at the injury site to form a fibrin clot which thus controls homeostasis.
The inflammatory phase is mainly characterized by removal of bacteria and debris, and factors released causing migration and cell division involved in the proliferative phase. After the wound occurrence, the process of extravasation of blood begins to fill in the inflicted area with plasma and cell elements, especially platelets. Platelet aggregation and blood coagulation generate a fibrin rich buffer which restores hemostasis and forms a barrier against the invasion of microorganisms and organizes a temporary matrix required for migration of cells. This matrix also serves as a cytokine and growth factors reservoir which is then used to release during the next phases of the healing process .Platelets are essential to the formation of hemostatic buffer and also secrete multiple mediators, including growth factors, released in the wound inflicted area. Platelets are induced by thrombin and release many growth factors, such as platelet derived growth-factor (PDGF), transforming growth factor beta (TGF-Î²), epidermal growth factor (EGF), transforming growth factor alpha (TGF-Î±), and vascular endothelial growth factor (VEGF), in addition to certain glucoproteins which are adhesive in nature such as fibronectin and thrombospondin. They are key ingredients of the transient extracellular matrix . The numerous chemotactic and vasoactive factors which support the recruitment of Inflammatory cells at the wound infliction site are produced by a combination of activation of a cascade of coagulation and the complement with the release of growth factors .
In the proliferative phase, there is the process of angiogenesis, deposition of collagen, formation of granulates, epithelialization and contraction of wound . By excretion of collagen, fibronectin and a formation of a new provisional extracellular matrix by fibroblasts the whole process of fibroplasia and tissue granulation takes place  .In the contraction phase the fibroblasts differentiate further into myofibroblasts, causing the wound to contract, in order to establish a grip on the wound edges thus contracting themselves using a mechanism similar to that in smooth muscle cells. In the Epithelial proliferation phase, in case of the ski begins by mitogenic and chemotactic stimulation of keratinocytes by TGF-Î² and EGF. Epithelialization is as important as the formation of granulation tissue and it starts at the repair process phase. However, before explaining angiogenesis, it is imperative to emphasize that the increased microvascular permeability is an important phase of this process allowing formation of temporary extracellular matrix, by leak of proteins, cytokines and cell elements which are necessary to migration and proliferation of endothelial cells ]. With the remodeling phase there is remodeling and realignment of collagen along tension lines and as the function of the cells nears completion, the unrequited cells undergo programmed cell death (Apoptosis). Thus the effect of Semaphorins and Plexins on vascular remodeling and regression can be closely assessed in relation to these three phases of wound healing. The final efficiency of this whole process lies with the fact that this process is not only a complex mechanism but is also susceptible to certain factors that may cause interruption or even failure thus leading to long term chronic non-healing wounds. Factors that mainly contribute to affecting this process include: diabetes, arterial disease, ageing, and infection . Increase in vascular permeability is preceded by formation of new blood vessels from the existing blood vessels . Formation of edema is directly in relation to the increase in vascular permeability to water and other macromolecules present. Such increases in permeability of the capillaries may seem to have a mild effect during physiological angiogenesis, but considerable damage is caused in some specific pathologies like diabetic retinopathy .
Remodeling phase is the final phase of the Wound Healing process. In this phase there is an attempt to recover to the normal tissue structure. This phase is mainly characterized by proteogylcan and collagen deposits by maturation and affection to extracellular matrix. Later ahead in this phase, there is a transformation of myofibroblasts from fibroblasts thus behaving as a contractile tissue responding to agonist that stimulate the smooth muscles. During the same phase of time, the extracellular matrix is reorganized which thus transforms the transient matrix into a definitive one. When observed in scars its phenotypic intensity also reflects the phenomenon that has occurred . As a result of maturation and remodeling processes, through a process of migration, apoptosis or other unknown death cell mechanisms, most vessels, fibroblasts and inflammatory cells disappear from the wound site, which leads to scar which has fewer cells.
Microvascular Endothelial Cells form the interior surface lining in the blood vessel and they are the key involved in angiogenesis and wound repair. They initiate an angiogenic process with response to injury which consists of microvascular hyperpermeability induction, local basement membrane degradation and then reconstruction, migration and sprouting into local stroma. It also consists of proliferation of cells, granulate tissue formation of new blood vessels and thus regression and involution of the newly formed vasculature as issue remodeling (Marx et al.,1994). It has been postulated that the proliferation of endothelial cells is a secondary effect of cell migration, therefore fibronectin, heparin and platlet factors which are known to stimulate endothelial cell migration into the wound also directly or indirectly stimulate cell proliferation .
Angiogenesis is crucial to the wound repair process, it is the growth of new blood vessels from the existing host vasculature (Folkman and Shing, 1992). It is a physiological process that involves the sprouting of new blood vessels from existing ones and is thus a crucial component of wound repair after maturation and also organ development during embryogenesis. Any sort of persisting imbalance leads in this process can lead to unregulated angiogenesis and is the pathogenic basis for a lot of diseases such as rheumatoid arthritis, muscular degeneration, and proliferative retinopathy etc. The main function of angiogenesis is to provide nutrition and oxygen to the growing tissue . With the exception of hair cycle and female reproductive cycle of the uterus and the ovaries, the vasculature remains quiescent but has the capacity to be able to initiate angiogenesis during wound repair. Over the past 20 years our understanding of the whole process of angiogenesis involved in wound repair has been greatly enhanced by advances in molecular and cellular biology . With response to cell injury it is dynamic process regulated mainly by pro and anti-angiogenic proteins released by the cells in the serum and the surrounding extracellular matrix (Risau, 1997). Based on the characterization of the three phases of wound healing, angiogenesis is thought to occur during the proliferation phase, though in fact, many of the signals of angiogenesis occur during the inflammatory phase.
Angiogenesis is known to be stimulated in the tissue repair process by a number of growth factors.Vascular endothelial growth Factor (VEGF), angiopoietin, fibroblast growth factor (FGF), Hepatocyte growth factor (HGF) & transforming growth factor beta are the major potent cytokines in wound angiogenesis . Some vessels survive and are stabilized by recruiting vascular smooth muscle cells and pericytes, while other vessels which are not needed undergo apoptosis.Work done in earlier groups has shown that endothelial progenitor cells (EPC) are involved early in this process of wound healing, although it is not clear that which factors regulate the EPC environment
Vascular smooth muscle cells are particular type of smooth muscle cells which comprise of majority of the walls of the blood vessels. They control the blood flow volume and pressure to the required areas with less oxygen. So their main function is to regulate and maintain the caliber of blood vessels in the body by contracting and expanding. They are recruited at the end of angiogenesis to stabilize the vessel wall. They are involved in remodeling vessels and vascular maturation in wound healing and therefore may also express neuropilins and plexins to regulate the final stages of wound healing.
VEGF is a disulphide-linked homodimeric, heparin-binding signal glycoprotein which is produced by cells in order to stimulate the growth of new blood vessels. Out of the major isoforms of VEGF (VEGF121, VEGF165, VEGF189 and VEGF206), there are at least six other isoforms of 121,145,165,189,206 amino acids which are generated by alternative splicing of a single gene (Tischer et al., 1991; Houck et al., 1991; Poltorak et al., 1997; Jingjing et al., 1999). Though these isoforms exhibit similar biological properties but they are different in their binding affinities to heparin and to the extracellular matrix (Park et al., 1993: Neufeld et al., 1999). Out of these isoforms, the larger ones remains associated with cells before activation by proteolysis and have transmembrane domains, while the smaller ones are secreted in soluble form. VEGF is known to bond to two types of endothelial cell specific type 3 receptors tyrosine kinase receptors, VEGF-R1 and VEGF-R2 (de Vries et al., 1992; Millauer et al., 1993). Several other proteins of the VEGF family whose function specifically in the process of wound angiogenesis in not yet determined completely have been identified which include VEGF-B, VEGF-C, VEGF-D, VEGF-E (Ferrara, 2001). VEGF-A is one of the most important members of this family which include other members aforementioned and which include Placenta Growth Factor (PIGF). Initially only the discovery of VEGF-A came into existence and thus it was called just VEGF. Functions of VEGF-A specific to angiogenesis involve migration of endothelial cells, mitosis of endothelial cells, methane-monooxygenase activity, AlphaV Beta3 activity and creation of blood vessel lumen. It also promotes vasodilation indirectly by release of Nitric Oxide. VEGF-A was originally referred to as vascular permeability factor. VEGF-B is known to promote embryonic angiogenesis. VEGF-C and VEGF-D have their main functions as lymphangiogenesis and development of lymphatic vasculature. Placental Growth Factor is required for mainly for vasculogenesis but is also needed for angiogenesis during wound healing and cancer. VEGF is known to stimulate the process of angiogenesis during tissue repair by regulating various processes including vascular permeability, migration & proliferation of existing endothelial cells and recruitment of marrow derived endothelial progenitor cells to the wound site (Keck et al., 1989). VEGF is upregulated in the early stages of wound healing and then gradually reduces after a few weeks but never to the normal pre-wound levels . VEGF may be crucial during the proliferation phase of granular tissue formation during wound repair for angiogenesis (Nissen et al. 1998). Thus on addition of the neutralizing anti-VEGF antibody to a healing wound, it strongly inhibits wound granulation tissue formation (Howdieshell et al., 2001). It has also been stated that tissue hypoxia during injury is a major inducer of this growth factor (Detmar et al., 1997). VEGF production is induced in cells which are not receving enough amount of oxygen. On being deficient in oxygen cell produces a trabscription factor known as hypoxia-inducible factor (HIF) which stimulates the release of VEGF. Then the VEGF circulating binds to VEGF receptors on the endothelial cells which in turn thus trigger a tyrosine-kinase pathway leading to angiogenesis.
VEGF-R1 & VEGF-R2 are two receptor tyrosine kinases which mediate the endothelial effects of VEGF. VEGF-A binds to VEGF-R1 and VEGF-R2. VEGF receptors have an intracellular portion containing a split tyrosine kinase domain, a single transmembrane spanning region and also and extracellular domain containing seven immunoglobulin like domains. Immunohistochemistry has shown that VEGF-R1 expression goes down during the angiogenic phase of wound healing and it appears to inversely regulate VEGF activity whereas VEGF R2 mediates growth and permeability actions and is upregulated during the angiogenesis phase of wound healing .
Neuropilins (Np-1 & Np-2) have recently been identified as VEGF binding receptors which are also expressed on endothelial cells . Neuropilins are transmembrane glycoproteins which were originally identified as being involved in neuronal development . They have been suggested to play a key role in angiogenesis . They are multifunctional non-tyrosine kinase receptors that bind to class 3 semaphorins and vascular endothelial growth factor. NRP-1 and NRP-2 interact with class 3 Semaphorins and were first identified for their key role in mediating axonal guidance in the development of the nervous system. There is growing evidence that supports the fact that there is a critical role for these receptors in progression of tumor cells. Neuropilin expression is known to be up-regulated in many tumor types, and is correlated with tumor progression and prognosis in some specific tumors. Neuropilins may also mediate the effects on tumor progression indirectly by affecting angiogenesis or by affecting tumor cells directly . IHC studies in humans have shown that the expression of Np1 and Np2 is restricted to endothelial cells with occasional occurrence in macrophages in healing wounds. Also an increase in Np1 endothelial expression was seen within a week after wounding, just before maximum wound vascularity & it remains elevated during the vascular regression phase . Np2 expression was observed in the angiogenic stages of the wound healing process following a similar pattern to Microvascular Density (MVD) & VEFG-R2  .Studies have shown that Np2 expression is restricted to endothelial cells but, in case of scar tissue it was occasionally found on Macrophages . In studies carried out recently, it was found out that in mice with NRP-1 knockout had disorganization of nerve trajectories as well as limbs and were not properly innervated . On the other hand, some other studies suggested that mice with NRP-2 knockout exhibit certain defects in development of spinal sensory axons as well as cranial nerves. They were also found to have a disorganized and even missing fiber tracts in the adult brain . The process of axonal repulsion which is quite necessary in axonal guidance is inhibited by neutralization of NRP-1 with blocking antibodies [29-32]. The effects of neuropilins on the nervous system are mediated by binding to a family of ligands known as Semaphorins.
Semaphorins are a class of secreted and membrane proteins that act as axonal growth cone guidance molecules by binding to neuropilins & their co-receptors the plexins. Each semaphorin molecule is characterized by the expression of a specific region of about 500 amino acids known as the sema domain. Different Semaphorins use different receptors to bind. Most Semaphorins use a group of proteins know as Plexins to bind while class 3 Semaphorins utilize a group of proteins known as Neuropilins as co-receptors with plexins. Class 7 Semaphorins are known to bind with integrins as their receptorsThere are eight existing classes of Semaphorins, out of which only class 3 semaphorins bind to neuropilins. In the category of class 3 semaphorins that range from Sema3A-Sema3F, those known to be able to successfully bind to neuropilins till date are Sema3A, Sema3B, and Sema3F and they exhibit antitumor properties. Sema3A has been found to slow down tumor cell chemotaxis . Sema3B and Sema3F are tumor suppressor genes and they lose their expression in lung cancers . Sema3F on the other hand is known to have antiangiogenic properties that contribute to antitumor effects it produces . Sema3A, Sema3B, and Sema3F do compete with VEGF to bind with neuropilin and they seem to be mutually antagonistic as well [37-41]. Also soluble Neuropilin-1 may act as an antagonist to VEGF . . Semaphorins and their receptors may be involved in the sorting of pools of motor neurons and also the modulation of pathfinding for afferent and efferent axons from and to these pools .Work in the lab has shown that semaphorin 3B is expressed during the vascular regression phase of human wound healing using Immunohistochemistry (Dr. CA Staton's unpublished observation).
Studies have also shown that class 3 Semaphorins are also secreted some cells types including tumor cells, where they perform an inhibitory effect in growth of tumor cells and angiogenesis (specifically Sema3B and Sema3F). Class three semaphorins with help of neuropilins and plexins primarily collapse the actin cytoskeleton thus inhibiting the cell motility and migration of tumor and endothelial cells. Besides binding to Class 3 semaphorins, neuropilin is known to bind with the protumorigenic and proangiogenic ligand vascular endothelial growth factor (VEGF). Recent studies attribute the antitumorigenic and antiangiogenic properties of class 3 semaphorins to compete with VEGF to bind with neuropilin receptors, on the other hand several others depict that class 3 semaphorins display growth-inhibitory activity independent of any competition with VEGF . Thus a further understanding of these molecular interactions and the role and signaling of class 3 Semaphorins in tumor biology will help us to determine whether class 3 semaphorins can represent as potential therapeutic agents in the field of modern day medicine.
Plexins are a family of proteins which acts as receptors for Semaphorins. Class 3 Semaphorins bind to both neuropilins & plexins with plexins mediating the signaling of this complex. Primarily cellular consequence of semaphorin signaling which is mediated by plexins is cytoskeletal collapse. These plexins are part of the Sema3-neuropilin ligand-receptor complex . Plexins are large transmembrane receptors with highly conserved cytoplasmic domains. The family of plexins consists of nine types, out of which four are type A plexins (A1, A2, A3, and A4), three are type B plexins (B1, B2, and B3), and two plexins C1 and D1 . Class 3 Semaphorins mediate their actions either via plexins type A or D1, which are known to affect actin depolymerization by employing a distinct GTPase-activating intracellular domain. It was first showed by Miao et al. that Sema3A causes retraction of endothelial cell lamellipodia by binding to endothelial NRP1 and inhibiting endothelial cell migration and capillary sprouting . In addition it was shown by a while ago (Serini et al. 2003) that class 3 semaphorins were capable to inhibiting the phenomenon of integrin activation on endothelial cells which is requisite for endothelial cells to be able to adhere to components of extracellular matrix to migrate . The inhibitory effects of class 3 semaphorins on integrins do seem to be dependent on plexins as well . As of now there are no clear mechanism that have been explained for plexin-mediated inhibition of tumor growth as well as angiogenesis, it is thus accepted universally that class three semaphorins use pelxins to mediate their actions to inhibit cellular motility and migration.
Thus it can be seen that angiogenesis is a highly ordered sequence of cellular events in response to tissue injury and is of crucial importance to the process of wound healing. Over time newly formed vessels mature and regress leaving a vascular scar and the mechanisms by which this process of vascular regression occurs is largely uncharacterized. It has been shown that Neuropilin-1 which is a co-receptor for proteins which stimulate angiogenesis (Including VEGF, FGF, HGF) and those though to inhibit angiogenesis( Class 3 semaphorins ), is elevated in all the wounds, including those undergoing vascular remodeling and regression . Therefore it is likely that Neuropilin, its semaphorin ligands and co-receptors, the plexins are involved in the vascular regression phase required for complete healing. As work in the lab has shown that Semaphorin 3B is expressed alongside neuropilin in the vascular regression phase of wound healing we hypothesise that " Semaphorin 3B modulates vascular regression via binding to neuropilin-1 and plexins on endothelial and vascular smooth muscle cells". Therefore the main aim would is to characterize the expression of Plexins, Semaphorin 3B and Neuroplins on vacular smooth muscle cells and microvascualr endothelial cells and also to assess the functional effects of Semaphorin 3B on endothelial cells and vascular smooth muscle cells. If time permits depending upon the rate at which we discover and analyze these results we will also try to assess signaling of semaphoring 3B through neuropilin and plexin.
Initially RT-PCR and Western Blot Analysis will be undertaken to establish which neuropilin and plexin receptors are present on endothelial, vascular smooth muscle and endothelial progenitor cells. Then the effects of semaphorins on cell proliferation, apoptosis, migration and the phosporylation/activation status of the receptors will be assessed.
Reverse transcription polymerase chain reactionÂ (RT-PCR) is type of a variation ofÂ polymerase chain reactionÂ (PCR). It is aÂ laboratory technique which is commonly used inÂ the field of molecular biologyÂ to generate millions of copies of a sequence ofÂ DNAÂ . In RT-PCR using the enzyme reverse transcriptase an RNA strand is firstÂ reverse transcribedÂ into itsÂ DNAÂ compliment (cDNA) and the resulting cDNA is amplified using traditional PCR orÂ real-time PCR. The high amount of amplification via RT-PCR provides a highly sensitive technique by which a very low copy number of RNA molecules can be detected. The only drawback of this techniques is that it is purely quantitative does not enlighten us about the location of proteins or their functions.
Western Blot Analysis also known as protein immunoblot is used to detect specific proteins on a given sample. It uses the technique of Gel Electrophoresis to separate the denatured proteins by the 3 dimensional structures of proteins. The proteins are further transferred to a nitrocellulose membrane where they are detected using specific antibodies targeted to the desired proteins . Assessment of signaling of semaphoring 3B through Neuropilin-1 and plexins will by using Western Blot Analysis for phosporylated signaling protein and receptors.
Immunohisochemistry is the process of detecting antigens like proteins for example in a cell or a tissue by exploitation of the biological principle that antibodies bind to specific antigens in a biological tissue . It is mainly used for diagnosis of cells which are cancerous such as tumor cells. But this technique brings its own drawback as it just tells us about the distribution and localization of differentially expressed proteins and not their quantity and functions.
To assess the functional effect of semaphoring 3B on endothelial cells and vascular smooth muscle cells we will use MTS assay for proliferation. Apoptosis will be done using Annexin V staining and Flow Cytometry. Migration of cells will be assessed by scratch assay and trans wells. Annexin 5 staining is done to quantify the number of cells undergoing apoptosis. In this assay we use a protein annexin 5 to tag to the apoptotic and dead cells and the number can be counted later with the help of flow cytometry or a fluorescence microscope. It can also be used as non-invasive way for detection of diseased tissue by using a radioactive labeled annexin AV.
Flow Cytometry is a techniques used for examination of small particles of the microscopic range. It is done by suspending the particles in a stream of fluid and then detecting them by passing through an electronic detection apparatus.
Future Prospects/ Conclusion
This project will enable us to identify the expression of Sema 3B and its receptors on Endothelial cells and Vascular smooth muscle cells and to establish the functional effects of Sema 3B an these cells, aiding in our comprehension of the process of Vascular Regression. Since almost all focused on the vascular initiation phase and not the remodeling or vascular regression phase, a better and more comprehensive knowledge about the vascular regression phase and normal wound healing may allow the development of more efficient and improved therapeutic strategies for abnormal wound healing.