A common thread in all forms of vascular disease, including cardiovascular disease, pulmonary arterial hypertension (PAH), aneurysms, and atherosclerosis, is the presence of low-grade inflammation that results in vascular and tissue remodeling. This inflammatory state arises due to infiltrating immune cells (leukocytes) and activated vascular cells such as endothelial cells (ECs), smooth muscle cells (SMCs), and adventitial fibroblasts (AFBs), all of which release growth factors and cytokines to trigger remodeling and further enhance the inflammatory state (creating a sustained feedback loop). The cause of this activation can depend on context and is for the most part, unknown. However, one of the most common signaling molecules that mediates this state of events is PDGF. In an article in Molecular Aspects of Medicine, Folestad et al. review and discuss the role of PDGFs in vascular pathologies, with a focus on the PDGF-C and PDGF-D isoforms.1
What is PDGF?
PDGF stands for “Platelet-derived growth factor (PDGF)”, and is one of many growth factors that regulate cell growth and division. It is a potent mitogen for cells of vascular origin (ECs, SMCs, and Fibroblasts).
There are four different types of PDGF molecules (PDGF-A, -B, -C, and -D), and these subtypes dimerize (bind together) with another unit before binding to the receptor (PDGFR). The receptors for PDGF are PDGFR-α and PDGFR-β, and these two receptors must dimerize to bind the PDGF dimers. Once bound, cell signaling cascades are induced within the cell. Here are the rules so far known for PDGF dimerization and binding to PDGF receptor dimers (x indicates the receptor dimer that the corresponding PGF dimer can bind to):
PDGF-A and PDGF-B signaling has been studied for some time, whereas PDGF-C and PDGF-D were recently discovered (in the early 2000s). All PDGFs are proteolytically cleaved in order to become active. PDGF-A and -B undergo intracellular proteolytic activation by furin, whereas PDGF-C and -D are proteolytically cleaved in the extracellular space, after secretion. PDGF-C is cleaved by the serine proteases plasmin and tissue plasminogen activator (tPA), while plasmin, urokinase plasminogen activator (uPA) and matriptase cleave PDGF-D. Thus, PDGF-C and PDGF-D rely on serine proteases from the fibrinolysis pathway for activation.
PDGF receptor expression varies widely. PDGFs on the other hand, are released primarily by platelets, macrophages, and vascular cells. They act in a paracrine manner, influencing the cells next to them, or in an autocrine manner, where a cell releases PDGF and PDGF, in turn, influences that same cell that released it. PDGFs and VEGFs are apparently interchangeable, in the sense that both have been found to bind to each others receptor (PDGF can bind to VEGF receptor, and VEGF can bind to PDGF receptor).
As with most molecules in the body, PDGF-C and -D play a role in both physiological and pathophysiological conditions. Specifically, PDGF-C is both of leukocyte and vascular origin, and is involved in, or associated with: 1) migration and proliferation of macrophages, ECs, and SMCs, 2) embryonic development, 3) fibrosis, 4) wound healing, and 5) angiogenesis. PDGF-D is primarily of vascular origin (ECs and SMCs), and is involved in, or associated with: 1) wound healing, 2) fibrosis, 3) cancer, and 4) cardiovascular disease.
PDGFRs are expressed broadly in cells of mesenchymal origin, which means cells that develop into connective tissue, blood vessels, and lymphatic tissue. Regarding tissue expression, under physiological conditions, PDGFR-α and PDGFR-β RNA are expressed virtually in all tissues, however, actual protein expression appears to be limited to brain, immune, kidney, GI, and adipose tissues. No protein expression occurs in the lung tissue, although this could change under pathophysiological conditions.
PDGFR-α appears to be critical for proper organ development, and for organ development processes. PDGFR-β appears to be critical for proper development of the vasculature: “PDGFR-β is mainly expressed by pericytes and its signaling is necessary for proper recruitment of vascular SMC and pericytes (Leveen et al., 1994). PDGF receptor activation has been particularly linked to angiogenesis where PDGFR-β expression especially correlates with increased vascularity and maturation of the vascular wall.” In animal models, deletion of either PDGFR-α or -β results in death in embryonic stages of development.
Dysregulation of PDGF receptor activity is observed in a number of pathologies. Specifically, PDGFR-β is strongly associated with vascular pathologies, while PDGF-α is associated with fibroblast and connective tissue pathologies, as well as fibrotic processes. Overall, under pathological conditions, PDGFR expression appears to increase. This increase primarily affects SMCs and fibroblasts. In response to increased expression and signaling, SMCs and fibroblasts “increase in proliferation, differentiation, apoptosis, migration and invasion, which promotes vessel wall pathologies and fibrotic tissue scarring.”
PDGFRs in Vascular Pathologies
During atherosclerosis, PDGFR-β is more abundantly expressed than the alpha receptor. This makes sense since, as mentioned above, PDGFR-β plays a role in anything vascular related, either vascular development or vascular pathologies. It appears to be critical in leukocyte migration during atherosclerosis and is present in fatty streaks. It is also associated with SMC activation, and SMC phenotype switching from contractile to synthetic dedifferentiated phenotypes. Also of note, low-density lipoprotein receptor-related protein 1 (LRP-1) can form a complex with PDGFR-β and activate this receptor.
With regards to cerebrovascular complications, such as a stroke, “activation of PDGFR-α on perivascular astrocytes is involved in the acute opening of the blood-brain barrier (BBB). In a study it was shown that treatment of mice with the PDGFR antagonist imatinib after an ischemic stroke reduced brain damage by decreasing the cerebrovascular permeability and hemorrhagic complications.” PDGFR-β appears to play a role in mediation and repair of cerebral tissue and the blood-brain barrier.
A few other facts:
- Excessive expression of PDGFR-β can activate SMCs, causing them to migrate to the intima of the blood vessel, proliferate, and create neointimal hyperplasia.
- PDGFR-β accelerates the growth of aortic vascular SMCs
- PDGFR-β drives adipose tissue neovascularization
PDGFs play a critical role during embryonic development, and early growth, but expression and activity in the healthy adult is low, and there is limited evidence of PDGF playing a role in normal physiological functions. The main purpose of PDGF in the healthy adult appears to be its role in wound healing and tissue repair. PDGF mainly acts with TGF-β to mediate tissue repair. It appears that, in wound healing, PDGF “is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-β, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.”2
PDGFs, specifically A and B, play a big role in phenotype switching of fibroblasts to myofibroblasts. Myofibroblasts are involved in the excessive extracellular matrix deposition observed in fibrosis. During fibrosis, TGF-β is also overexpressed, and this also promotes the formation of myofibroblasts. TGF-β and PDGF interact with each other through a PDGF-TGF-β signaling axis. PDGF and TGF-β expression and signaling have been shown to increase during fibrosis, however, the exact roles of each are unclear. For example, some studies show upregulation of PDGF-D during fibrosis, while others show downregulation.
As with all things, balance may be needed: “Overexpression of PDGF-C could contribute to fibrosis formation, whereas low expression of this growth factor could be involved in delayed wound healing (Eitner et al., 2008; Glim et al., 2013). In line with these results, increased wound healing was observed in diabetic mice when PDGF-CC was applied (Gilbertson et al., 2001). Diabetes mellitus is one of the leading causes of impaired wound healing. A study on diabetic rats demonstrates that a certain expression level of PDGF is necessary for normal tissue repair (Li et al., 2008).”
During cardiac infarction, PDGF-D appears to be upregulated, whereas PDGF-B and C appear to be reduced. Overexpression of either C or D can cause cardiac fibrosis and resulting dilated cardiomyopathy.
PDGF-D is also produced by macrophages during atherosclerosis and is present in fatty streaks. In this context, PDGF-D inhibits the expression of α-SMA and other proteins critical for maintaining the contractile phenotype of SMCs. PDGF-D is involved in the formation of neointimal hyperplasia after vascular injury.
PDGFs also play a role in neovascularization processes. Aside from physiological necessity, neovascularization is important for creating new blood vessels during ischemia, however, it is probably detrimental during tumorigenesis. PDGF-Cs are upregulated in ischemic heart blood vessels. They are also upregulated in all angiogenic tissues such as tumors (inhibition of PDGF-C can block tumor growth), the placenta, ovary and embryonic tissues.
Following vascular injury, platelets hone to the site of injury and are responsible for the recruitment of endothelial progenitor cells, which in turn are responsible for neovascularization processes. The platelets release PDGF-C, which is able to stimulate the recruitment of endothelial progenitor cells to the site of vascular injury. Actually, both PDGF-C and -D participate in this processes. Some evidence in animal models suggests that dysregulation of PDGF-C can contribute to impaired angiogenesis.
PDGF-D promotes angiogenesis via activation of Notch-1 and NF-kB, which then goes on to activate VEGF pathways and MMP-9 activity.
Animal Model Studies of PDGFs/PDGFRs
- PDGF-C knockout is either 1) lethal, or 2) presents with cerebral ventricular malformations, abnormal vascularization and skeletal deformations, depending on the genetic background.3
- Introducing PDGF-C knockout in a neurodegenerative vascular disease animal model (such as amyotrophic lateral sclerosis) restores vascular barrier properties and reduces motor neuron loss, thus implying that PDGF-C accelerates neurodegenerative vascular processes.
- In a unilateral ureteral obstruction animal model, lack of PDGF-C reduces renal fibrosis
- In a unilateral ureteral obstruction animal model, lack of PDGF-D reduces renal fibrosis
- Lack of PDGF-C impairs browning of white adipose tissue
- In a mouse model of pancreatic neuroendocrine tumors, lack of PDGF-D delayed tumor growth
- Overexpression of PDGF-C and PDGF-D in transgenic mouse models typically leads to fibrosis.
BOTTOM LINE: Lack of PDGF-C is NOT good, but too much PDGF-C is NOT good either.
PDGF & PDGFR in Pulmonary Hypertension
Not surprisingly, with regards to PH, both PDGF and PDGFR expression and activity is also upregulated. PDGF-a, PDGF-b, PDGFR-α, and PDGFR-β mRNA expression are all increased in the small pulmonary arteries of PH patients, with significant upregulation in PDGFR-b observed (compared to controls).4
PDGF-A and PDGF-B were mainly found in SMCs and ECs in the small pulmonary arteries of PH patients, whereas PDGFR-α and PDGFR-beta were mainly found in the SMCs of small pulmonary arteries.
PDGFR receptor blocking studies have shown promise in animal models and in humans, but have also caused severe side effects in humans, highlighting the dual role that PDGFRs play in both physiological and pathophysiological conditions.
Folestad et al. conclude by recognizing the importance of other factors in PDGF signaling, specifically, the proteases that cleave and activate PDGFs: “Several of the suggested functions of the novel PDGFs in different pathological vascular conditions come from studies reporting dysregulation of PDGF-C and PDGF-D where they are either up or down regulated. It might be that PDGF-C and PDGF-D do not need to be overexpressed to cause undesired cellular activation and pathological conditions. If the protease that can activate PDGF-C or PDGF-D is presented in an excess amount, it can cause increased activation of PDGF-C or PDGF-D, without any need for increased expression of the ligand itself. In order to accurately understand the role novel PDGFs play in different vascular pathological conditions, it is important to further study the endogenous factors involved in their regulation. The following description of the suggested role of PDGF-C in stroke illustrates why it is so important to understand the exact role of all the players involved in the biology of the novel PDGFs. The serine protease tPA, which is known to dissolve blood clots and to be involved in the activation of PDGF-C, is used to treat ischemic stroke patients. Thus, while tPA helps to dissolve the clot quickly and restore blood flow to the brain tissue it can also activate PDGF-C that is expressed in the brain, which enhances the opening of the blood-brain barrier. This allows an influx of inflammatory cells into the brain, contributing to edema, hemorrhagic transformation, and increased mortality. Treatment with tPA is the only FDA-approved treatment for stroke, but because of the effects caused by the opening of the blood-brain barrier, it must be started within 4.5 h of symptom onset. This limits the number of patients that can be treated with tPA. However, in animal studies, Su et al. found that blocking PDGFR signaling with imatinib reduced the neurotoxic effects of tPA and allowed later application of tPA after onset of stroke (Su et al., 2009). Furthermore, in the recent I-Stroke study by Wahlgren et al. it was suggested that imatinib is safe and generally well tolerated in ischemic stroke patients treated with tPA (Wahlgren et al., 2017). These findings not only enable better treatment of stroke patients, they also show how important it is to understand all the players involved in the biology of the novel PDGFs.”
- PDGFs and their receptors are critical for physiological purposes, but also play a role in disease
- PDGFRa is involved with organ development
- PDGFRb is involved with vascular development, and vascular pathologies
- PDGFs and VEGFs are apparently interchangeable, in the sense that both have been found to bind to each others receptor (PDGF can bind to VEGF receptor, and VEGF can bind to PDGF receptor). Dysregulation of PDGF-D appears to be more prevalent than PDGF-C in cardiovascular diseases
- PDGF-Cs appear to play a role in fibrotic processes. However, PDGF-Cs also play a critical role in tumor angiogenesis
- Too much PDGF/PDGFR signaling can be problematic, and too little PDGF/PDGFR signaling can be problematic
Other interesting observations & notes:
- “Interestingly, recent findings show that PDGFR signaling can be modified by neuropilin-1, which binds PDGF-D and functions as a co-receptor in PDGF-D/PDGFR-β signaling (Muhl et al., 2017).” Neurolipin-1 is a co-receptor for VEGF, and thus making it a player in angiogenesis processes. Thus, PDGF-D may play a role in angiogenesis.5 Also, interestingly, the authors assert that this “suggests that PDGF-D binding to NRP1 could change the availability of NRP1 for other endothelial signaling pathways, such as VEFG-A-VEGFR-2 signaling.”
- PDGF-C is released by endothelial cells during white adipose tissue angiogenesis and regulates the conversion of white adipose tissue to brown adipose tissue
- In transgenic mouse models with over-expression of either PDGF-C or PDGF-D, researchers found that “several vascular complications such as vascular remodeling, including dilation of vessels, increased density of SMC-coated vessels, and proliferation of vascular SMCs, leading to a thickening of tunica media was seen when PDGF-D was overexpressed. Similar vascular abnormalities were seen overexpressing PDGF-C but the thickening of arterial walls was a unique feature induced by PDGF-D overexpression.”
- The pathways through which PDGF signaling takes place may depend on context: “Autocrine activation of PDGF signaling pathways is involved in certain gliomas, sarcomas, and leukemias. Paracrine PDGF signaling is commonly observed in epithelial cancers, where it triggers stromal recruitment and may be involved in epithelial–mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis.”6
- Both PDGF/PDGFR and TGF/TGFR pathways play a critical role in physiology as well as disease. Therefore, understanding the complex and nuanced PDGF/PDGFR and TGF/TGFR pathways from a systems biology (big data, machine learning approach) would be key to solving fibrosis and vascular diseases.
- PDGFRs may be a critical factor in PAH, but not necessarily a cause of PAH. Or, if it is a cause, then care must be taken to only target overactivated receptors, as blocking of all PDGFRs may lead to detrimental effects. Also, remember that PDGFRs appear to be underexpressed in lung tissue during physiological conditions. Thus, perhaps we should look to targeting lung tissue specific PDGFRs, and not necessarily vascular cell PDGFRs? Perhaps lung tissue specific PDGFRs that become expressed during PAH processes could be a target?
- What if PDGF-D binds to neurolipin and prevents VEGF binding? This would be a causative factor for PH if PH is an apoptosis driven condition such as in SUGEN/Hypoxia animal model.
- What is the role of PDGF-C and PDGF-D, if any, in Pulmonary Hypertension?