Various animal models have been used to study the effects of perlecan. In 1999, both Costell and Arikawa-Hirasawa et al. developed perlecan knockout mice models (Arikawa-Hirasawa et al., 1999; Costell et al., 1999). Costell et al. developed perlecan knockout (KO) mice (i.e., Hspg2^−/−) by removing exon 6 of the perlecan gene (Costell et al., 1999). Such homozygous knockout models resulted in chondrodysplasia, cleft palate, microaneurysms, and hemopericardium. Further examination of these models posited that inability to withstand stress on developing cartilage and cardiac basement membranes led to respiratory failure and fatality soon after birth (Costell et al., 1999). Arikawa-Hirasawa et al. removed exon 7 of the HSPG2 gene, resulting in homozygous mutant mice that were verified to demonstrate no perlecan expression via northern blot and immunoprecipitation (Arikawa-Hirasawa et al., 1999). These models also displayed embryonic death, chondrodysplasia, and skeletal abnormalities (Arikawa-Hirasawa et al., 1999). Perlecan animal models and subsequent biologic effects are depicted in Figure 1.

Xu et al. (2016) first utilized tissue-dependent conditional animal models of perlecan via the creation of knockout mice that lacked perlecan in skeletal muscle, yet restored perlecan expression in cartilage via a perinatal-lethality rescue mechanism involving the Col2a1 promoter and enhancer, which successfully allowed tissue-dependent roles of perlecan to be explored. This model has since been used to study perlecan’s specific role in neurogenesis, (Kerever et al., 2014), vascularization of bone (Ishijima et al., 2012), and maintenance of the blood brain barrier (Nakamura et al., 2019).

Perlecan has been established as a proteoglycan critical for cartilaginous and chondrocyte formation in the developing fetus (Arikawa-Hirasawa et al., 1999; Costell et al., 1999). Perlecan surrounds stem cell niches in rudimentary cartilage, as seen in Table 1 ²².There, the molecule acts as a signal for FGF-2 and FGF-18 induced migration and coordination of chondroprogenitor stem cells necessary to effectively cavitate rudimentary joints. FGF-2 allows mesenchymal stem cells to adopt the chondrogenic phenotype (Shu et al., 2016). Furthermore, the differentiation and hypertrophy of such chondrocytes allows ossification centers to form via FGF-18 induced osteogenesis. Once mineralization occurs, Perlecan is critical in establishing homeostasis and maintenance of permanent cartilage by regulating FGF-2 and FGF-18 growth factor bioavailability (Shu et al., 2016). This allows cartilage to slowly renew while maintaining the structural integrity given to it by FGF-18 induced structurally sound columnar chondrocyte arrangements within the growth plate. Additionally, HSPG-2 knockout mice were found to have defective endochondral ossification with disorganized collagen fibrils (Arikawa-Hirasawa et al., 1999) and chondrocyte columnar structures. Additionally, perlecan-null mice demonstrated decreased chondrocyte proliferation and dyssegmental ossification of the spine (Arikawa-Hirasawa et al., 2001). Two diseases arise from frameshift mutations within the HSPG-2 gene, including the heterozygous and milder chondrodysplasia, Schwartz-Jampel Syndrome, and the homozygous neonatal lethal disorder, Silverman-Handmaker Dyssegmental Dysplasia. The pathology of both diseases is contributed to by a functional diminishment or absence of perlecan and thus unstable extracellular matrix support in development (Arikawa-Hirasawa et al., 1999; Arikawa-Hirasawa et al., 2001).

Schwartz-Jampel Syndrome is an autosomal recessive disorder with one affected allele of the HSPG-2 gene that results in less than 10% of functional perlecan levels in the skeletal muscle, fibroblasts, heart and the kidney (Martinez et al., 2018). This disorder is characterized by growth plate disorganization and failed union of the chondro-osseous junction, resulting in features such as misshapen and shortened long bones, facial dimorphism, pigeon breast and myotonia (Costell et al., 1999; Stum et al., 2008; Martinez et al., 2018). Such clinical symptoms of patients with this disorder is depicted in Figure 2. Patients of this disorder typically survive without any alterations to life expectancy; however, the severity of disease is inversely correlated with the amount of functional perlecan secreted into the extracellular matrix in development (Stum et al., 2008).

Silverman-Handmaker Dyssegmental Dysplasia (DDSH) is the more severe form of the two perlecan associated chondrodysplasia disorders. DDSH arises from a frameshift mutation in both alleles of the HSPG-2 gene, resulting in premature termination and a truncated perlecan protein that undergoes proteolytic degradation (Arikawa-Hirasawa et al., 2001; Martinez et al., 2018). This results in a functionally null gene and absent perlecan secretion into the extracellular matrix, rendering outcomes of affected neonates as lethal. This autosomal recessive chondrodysplasia is characterized by anisospondyly, micromelia, flat facial features, encephalocele, and dysplastic ossification of the spine (Arikawa-Hirasawa et al., 2001). The complete lack of perlecan in this disorder also results in shortened growth plates, dyssegmental organization of growing bones, and degeneration of cartilage, likely from unstable extracellular matrix scaffolding required for cell organization (Arikawa-Hirasawa et al., 1999; Costell et al., 1999; Arikawa-Hirasawa et al., 2001).

In addition to diseases arising from diminished perlecan levels, the pathophysiology of sarcopenia and OA seem to involve increased, rather than decreased, perlecan expression. Sarcopenia is the process of age-related skeletal muscle atrophy and was determined to arise due to satellite cell apoptosis and erroneous capillary function (Wang et al., 2014). Due to perlecan and its C terminal fragment, endorepellin, acting via VEGFR2 on endothelial cells, Bechet et al. examined if either molecule was upregulated in sarcopenic models. Utilizing mouse gastrocnemius, it was found that there was increased apoptosis of capillary endothelial cells, expression of endorepellin, and ECM fibrosis (Wang et al., 2014) in aged specimens compared to controls. Endorepellin functions in an inhibitory manner on receptors crucial for angiogenesis, such as VEGFR1 and 2, and therefore may negatively impact skeletal muscle capillary health in aging individuals (Goyal et al., 2011; Wang et al., 2014; Douglass et al., 2015).

Perlecan is also found to be implicated in the pathophysiology of OA. OA affects approximately 27 million US adults and is characterized by deterioration of cartilage, synovitis, and osteophyte formation (National Collaborating Centre for Chronic Conditions, 2008; Kaneko et al., 2013). Such pathology results in stiffness and pain of the joints, making OA one of the leading causes of mobility impairment in elderly populations (National Collaborating Centre for Chronic Conditions, 2008). The work of Arikawa-Hirasawa et al. using perinatal lethality rescued perlecan-knockout mice (HSPG-2 ^−/−) examined perlecan’s role in forming osteophytes. In this model, perlecan is lacking in all synovial fluid and joint tissue besides cartilage. Arikawa-Hirasawa et al. utilized both surgical and TGF-B induced osteophyte formation methods to examine osteophyte characteristics. In both models, HSPG-2 knock-out mice had significantly reduced osteophyte size and maturation (Kaneko et al., 2013). This expands on the finding that primary synovial cells upregulate TGF-B and perlecan in patients with OA (Dodge et al., 1995). Thus, synovial perlecan may play an important role in osteophyte development and inhibition of such signaling pathways may serve as an effective target for OA therapeutics. Perlecan’s involvement and mechanism of action in the musculoskeletal diseases discussed is summarized in Table 2 and Figure 3.

Just as perlecan is necessary for osseous and cartilaginous function, it is also critical in the development and pathophysiology of neural systems. Perlecan’s side chains interact with FGF-2 within the neural stem cell niches of the sub-ventricular and sub-granular dentate gyrus of the hippocampus, effectively promoting stem cell self-renewal and neural propagation (Table 1) (Hayes et al., 2022). Furthermore, reduction of Perlecan’s signal transduction structure with FGF-2 in the sub-ventricular zone has been seen in aged models (Hayes et al., 2022). Such effect is thought to occur by diminished cell survival and proliferation via failure of FGF-2 induced phosphorylation of extracellular signal-regulated kinase (Erk1/2). Perlecan is also critical for proper cephalic development, as HSPG-2 ^−/− knockout mice experienced diminished development of the hindbrain and forebrain with death or exencephaly occurring shortly after fertilization (Arikawa-Hirasawa et al., 1999). Additionally, perlecan null mouse models experience collapsed brain vesicles, likely due to weakened basement membranes that cannot withstand the shear stress of developing neural structures of which perlecan can provide structural integrity (Arikawa-Hirasawa et al., 1999; Costell et al., 1999; Martinez et al., 2018). As such, functionally null models without perlecan are embryonic lethal due to weakened capillary walls and subsequent bleeding in the brain, lungs, and heart of the developing embryo (Martinez et al., 2018).

Due to its central role in neurodevelopment and angiogenesis, perlecan has been examined as a therapeutic for ischemic stroke. Stroke is the primary contributor to long-term disability in the United States (Roger et al., 2012). In addition, the only viable treatment remains tissue plasminogen activator (tPA), though this requires an incredibly narrow efficacious window of only 4.5 h post ischemic attack. Our recent work investigated the effects of domain V of perlecan as an effective broad therapy for ischemic stroke agent from previous findings that perlecan may be neuroprotective and that domain V is upregulated in the post-stroke human brain (Lee et al., 2011). We found that in stroke mouse models receiving domain V 7 days after ischemic event, peri-infarct excitatory synapse generation extended further into the neocortex compared to mice that did not receive domain V. Furthermore, perlecan deficient mice demonstrated less neuroblast precursor cells post-stroke than those with functional perlecan expression (Trout et al., 2021). Such neuro-reparative outcomes may function through the α2β1 integrin receptor signaling pathway, as blockade of this receptor mostly ameliorated the observed effects, although perlecan can bind to several other integrin receptors. Lastly, compared to control mice, mice who received perlecan domain V also experienced better neurological outcomes post ischemic stroke via regulation of pericyte migration. Mediating pericyte transport and facilitating connection to the extracellular matrix is critical for repair of the blood brain barrier that becomes “leaky” after ischemia (Roberts et al., 2012). Domain V administration recruited pericytes to the BBB via a PDGFR-β and integrin α5β1 signaling mechanism, and acted to regulate focal adhesion of extracellular matrix components and stabilized the actin cytoskeleton (Nakamura et al., 2019). Perlecan knock-out mice had a lower overall survival rate, along with increased weight loss and neuro-deficits compared to control mice (Nakamura et al., 2019). Perlecan’s role in restoring the blood brain barrier post stroke via pericyte modulation demonstrates its potential as an efficacious therapeutic. As domain V administration additionally enhanced synaptic neurogenesis and neural precursor cells in this model, this makes perlecan and its derivatives a promising potential therapy that warrants further investigation.

Altered levels of perlecan has also been associated with the pathophysiology of brain arteriovenous malformations (BAVM). BAVM is a grouping or “nidus” of dysplastic arteries and veins in the brain that lack a true capillary bed (Kim et al., 2009). Subsequently, they serve as a shunt that sends arterial blood into venous circulation, putting the individual at risk of intracranial hemorrhage and mortality (Kim et al., 2009). BAVMs are overly angiogenic via increased VEGF signaling, a pathway through which perlecan domain V participates in. In human BAVM specimens, perlecan’s endogenously cleaved segment, domain V, and its pro-angiogenic signaling molecule, α5β1 integrin, were both increased compared to control brain tissue (Kahle et al., 2012). Domain V levels were 14 times higher in the BAVM specimens, and VEGF and TGFβ were increased 9-fold compared to control (Kahle et al., 2012). This provides evidence that perlecan domain V’s upregulation of VEGF may be critical for formation of a BAVM nidus and that inhibition of this mechanism should be investigated further as a therapeutic target.

Perlecan’s angiogenic proclivity may also cause it to be a potential therapeutic target for the treatment of AD. AD remains a leading cause of disability and mortality in the United States, with resultant deaths increasing by 146% from 2000 to 2018 (Alzeimer’s Association, 2020). The disease is characterized by neurofibrillary tangles and misfolded deposits of amyloid-β (Aβ) of various sizes forming in the cerebrovasculature. This consequently results in damaged endothelial lining, decreased VEGF levels and angiogenesis, and overall diminished neurovascular status that promotes the hallmark symptoms of depression, confusion, loss of memory, and dementia (Parham et al., 2014; Alzeimer’s Association, 2020). Endothelial damage subsequently disrupts the blood brain barrier and therefore diminishes Aβ clearance pathways, resulting in neurotoxic buildups of Aβ (Parham et al., 2014; Nakamura et al., 2019). Experimental administration of domain V to AD mouse models effectively blocked the reduced cerebral endothelial cell proliferation that resulted from Aβ_25-35 administration (Parham et al., 2014). Administering perlecan domain V also restored the ability of mouse brain endothelial cells to begin forming “tube” like capillary structures even with the presence of Aβ_25-35. Such findings suggest perlecan domain V may outcompete the Aβ plaques for VEGFR2, and thus may restore angiogenesis and endothelial cell integrity to more functional and healthy levels. As domain V of perlecan is known to be pro-angiogenic through α5β1 integrin signaling and upregulation of VEGFR2, this may ultimately be the mechanism underlying its anti-angiogenic effects to Aβ and the way in which it provides protection to the cerebrovasculature (Aviezer et al., 1994). Experimental treatments in animal models using perlecan level manipulation are summarized in Table 3 and Figure 4.