title: In vitro effects of VEGF and PDGF on cells involved in spinal cord injury
Creators: name: Basu, Souptik
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Spinal cord injury (SCI)
subject: vascular endothelial growth factor (VEGF)
subject: platelet-derived growth factor (PDGF)
subject: cells
subject: host immune cells
subject: transplantable peripheral glial cells

description: Spinal cord injury (SCI) is a debilitating disease affecting 13.1-220 cases per million people around the world. The pathogenesis of SCI is complex and is orchestrated by a plethora of cellular and biochemical reactions. The initial phase (primary) is a transient one which occurs as a result of the initial impact and other related vertebral fractures. Subsequently, leakage of blood spinal cord barrier and apoptosis of neurons with spread of axonal/myelin debris become the key reasons that trigger a flare of immunological secondary responses. The initial cells to get activated are the macrophages/microglia (tissue-resident macrophages). Once activated they set off a cascade of harmful/congenial reactions which may further promote or reduce inflammation, respectively. Only an imbalance to this equilibrium can predispose the local environment to a catastrophic outcome. Hence, targeting these cells in the acute phase of SCI is seen as an ideal strategy to reduce inflammation and promote early recovery. However, no definite drug therapy exists for SCI treatment, at present. New avenues are still being explored for consideration. One such strategy that has been introduced in the last few years is the use of growth factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Both are well known angiogenic promoters and have neuroprotective activity. Hence, when a combined administration was initiated on rat SCI models, it was seen as an ideal therapeutic approach. However, the outcomes were inconsistent and needs an empirical dissection of the contributions and interactions of different cellular and molecular modulators of SCI. This thesis has thus been focused on the study of (1) the effect of the growth factors on the cells involved in modulation of acute secondary stage of SCI (macrophages, microglia, astrocytes and neurons) and (2) on transplantable peripheral glial cells (Schwann Cells-SCs and olfactory ensheathing cells-OECs) and the corresponding interaction of the latter with host myeloid cells (macrophages), in vitro. I have used macrophages and administered single and combined VEGF and PDGF to understand how transcription regulator dynamics (Akt) is affected under non-inflammatory and inflammatory conditions. Subsequently, I have also targeted the activation of macrophages through nuclear factor kappa B (NF-κB) protein translocation assays and determined the expression of M1 (pro-inflammatory) and M2 (anti-inflammatory) genes. My results showed that Akt overactivation by combined factors led to a downregulation of the protein. Overactivation was evident as the growth factors triggered significant NF-κB translocation to nucleus in inflammatory state. Additionally, administration of PDGF in inflammation restricted Cd163 (M2 gene) downregulation, showing that growth factors promoted the prevalence of anti-inflammatory macrophages in inflammatory stage. When growth factors were administered on microglia, the tissue resident macrophage, only PDGF was able to activate microglia (based on shape morphology), but this did not stimulate significantly higher expression of A1-inducing genes (Tnfa, Il1a and C1q). In fact, PDGF also stimulated reactive astrocytosis in inflammation through microglia. Additionally, both PDGF and combined VEGF+PDGF treated microglia in inflammation triggered better synaptosomal regulation by astrocytes. Although, the growth factors did not promote A2 (anti-inflammatory) gene expressions, the combined VEGF+PDGF was able to promote neuroprotection by restriction of TAR binding protein-43 (TDP-43) translocation, a stress granule formed in neurodegeneration, into cytoplasm and downregulation of neuron specific enolase enzyme as evident on RT-qPCR assay. In the last phase of my thesis, I studied the role of the growth factors on the modulation of transplantable peripheral glial cells like SCs and OECs. The growth factors (single and combination) promoted higher proliferation of SCs as was evident through p75NTR and EdU proliferation assay. Although, inflammation reduced the rate of clearance of myelin debris, PDGF was able to stimulate it, in both non-inflammatory and non-inflammatory conditions. Additionally, PDGF also reduced the TNF-α secretion (a pro-inflammatory cytokine) by SCs in inflammation. I also assessed the role of the growth factors on nerve repair associated and myelin expressing genes in SCs. Inflammation downregulated nerve repair associated genes Jun and Sox2. While combined VEGF+PDGF reduced Gdnf compared to PDGF, there was no obvious change in inflammation. However, for myelin expression, PDGF significantly downregulated Pou3f1 gene, in normal and inflammatory conditions. Thus, PDGF favoured the production of nerve repair over myelination in acute inflammatory condition. In the final part of my thesis, I studied the interaction of macrophages with OECs when growth factors were administered in inflammatory condition. I also checked for temporal progression of the modulation of OEC functionality, having treated the macrophages for early (3 h), middle (24 h) and longer (48 h) time points. Combined VEGF+PDGF showed variable progression of NF-κB translocation at the early time point, where there was no difference from inflammatory mediator to the middle time point where it was significantly lower, and finally at the longer time point where it was higher but overall protein intensity was low for all the groups (VEGF, PDGF and VEGF+PDGF). An added advantage from combined VEGF+PDGF was that it did not produce excess pro-inflammatory cytokine (TNF-α and IL-6) over inflammatory condition and maintained the phagocytic abilities of OECs to clear myelin debris. Interestingly, macrophage conditioned media treated for longer time point with combined VEGF+PDGF in inflammation helped OECs to express nerve repair associated genes. In conclusion, this thesis provided an in vitro understanding of VEGF and PDGF (as single and combined dose) interaction with cells involved (host immune cells and transplantable peripheral glial cells) in SCI. Due to the presence of a hostile microenvironment associated with presence of variable cues, use of any individual growth factor to achieve positive therapeutic outcome would be difficult. Hence, a combination would help to ameliorate the approach and provide a congenial outcome. However, further interaction studies would be necessary to unravel other beneficial effects of the growth factors to promote is as a viable therapeutic strategy.