Perlecan is a large multi-domain ECM HSPG detectable in four cell blastocysts during embryogenesis, its expression changes spatiotemporally during implantation of the embryo and in the placentation stage of embryonic development (Smith et al., 1997). Perlecan is expressed by many embryonic cell types and tissues (Roediger et al., 2009) including the trophoblast and tropho-ectoderm, the basal lamina that underlies the uterine epithelia and endothelia, and developing decidua (Smith et al., 1997). Perlecan promotes many biological processes in the embryo during cell adhesion, growth factor binding, and modulation of apoptotic events (Girós et al., 2007; Soulintzi and Zagris, 2007). Perlecan expression and activity is controlled at the transcriptional level, through alternative splicing, and proteolytic processing of perlecan in the extracellular environment (Gomes et al., 2002). Full length perlecan acts as an extracellular scaffold supporting cell attachment, growth factor and morphogen sequestration that promoting cell proliferation and differentiation, matrix production and tissue expansion during development (Farach-Carson and Carson, 2007). Perlecan also stabilises the ECM through multi-component interactions mediated by its core protein and its GAG chain substitution in domain I. Perlecan is a ubiquitous modular instructive multifunctional extracellular and pericellular PG that regulates cellular migration, differentiation and proliferation (McGrath et al., 2005; Whitelock et al., 2008; Lord et al., 2014; Nakamura et al., 2015b). Perlecan in cartilaginous tissues promotes proliferation and differentiation of chondrogenic cell types, stimulates matrix synthesis and contributes to tissue expansion and skeletogenesis. It also helps stabilize the extracellular matrix (ECM) and promotes various tissue repair processes (Costell et al., 1999; French et al., 2002; Gomes et al., 2004; Sadatsuki et al., 2017; Gao et al., 2021; Garcia et al., 2021). In vascular tissues, perlecan has different effects; for example, it promotes angiogenic repair of skin wounds through FGF-2 sequestered by its domain I HS chains (Zhou et al., 2004) but inhibits smooth muscle cell (SMC) proliferation and migration (Koyama et al., 1998; Gotha et al., 2014) (Figures 3, 4).

In a carotid artery injury model using MΔ3/Δ3 mice lacking perlecan HS chains the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased (Gotha et al., 2014). Perlecan lacking HS side chains has a reduced ability to inhibit SMC proliferation in vitro. The HS side chains of perlecan have critical roles to play in the vessel wall, as an interleukin (IL)-2 receptor for vascular SMCs (Arumugam et al., 2019). IL-2, a 15 kDa immunoregulatory cytokine secreted by T cells, promotes peripheral immune cell growth and development initiating a defensive immune response as an initial response in the wound repair process. While SMC proliferation is inhibited by HS-PGs, digestion of HS chains from perlecan reverses this effect. SMC perlecan is a hybrid HS/CS PG while endothelial perlecan is mono-substituted with HS (Whitelock et al., 2008; Lord et al., 2014; Melrose, 2020). SMCs bind to perlecan core protein only when perlecan’s GAG side chains have been removed. This involves a novel binding site in perlecan domain III, perlecan domain V and α2β1 integrin. Endothelial cells, however, adhere to perlecan core protein containing intact GAG substitution (Whitelock et al., 1999; Lord et al., 2014).

SMC perlecan binds FGF-1 and FGF-2 via HS side chain interactions promoting FGF-2, but not FGF-1 signaling (Lord et al., 2014). Endothelial cell perlecan also binds both FGF isoforms through HS but, in this case, promotes signaling through both FGF-1 and FGF-2. Perlecan differentially regulates cellular proliferation and cell signaling promoted by growth factors to regulate tissue repair processes (Segev et al., 2004; McGrath et al., 2005; Hultgårdh-Nilsson and Durbeej, 2007). The form of perlecan present in tissues can vary with different cell populations assembling variable GAG side chain components in domain I. Perlecan is also subject to post-translational modifications including truncations, mis-sense and point mutations in its core protein and proteolytic modifications leading to the generation of perlecan fragments (Melrose, 2020). Domain IV of full-length perlecan is particularly susceptible to cleavage by matrix metalloproteinases (MMPs) leading to the generation of bioactive domain I and V matricryptic fragments.

Attachment of perlecan to the endothelial cell surface as a dynamic flow sensor at the endothelium-blood interface (Siegel et al., 2014) can also modulate charge density at the endothelial cell surface affecting membrane polarization (Siegel et al., 2014). Shear stress signaling to endothelial cells regulates vascular ECM remodeling and induction of angiogenesis (Russo et al., 2020). Membrane polarization regulates cell proliferation, cell signaling, cytoskeletal organization and gene expression (Gradilla et al., 2018). Cell polarization facilitates cell-cell signalling and is interfaced with stimulatory biophysical forces at the cell surface that regulate cell differentiation and tissue development (Saha et al., 2018). Calcium signalling initiated through transient receptor potential (TRP) endothelial cell channels (Thakore and Earley, 2019) drives vasculogenesis and controls the contractile properties of SMCs, vasodilation and blood pressure (Thompson et al., 2011). Perlecan domain II is a LDL receptor that facilitates lipid clearance from the circulation (Ebara et al., 2000). Membrane de-polymerization resulting from binding of LDL to perlecan leads to vasoconstriction, lowering of cyclic guanosine monophosphate (cGMP) SMC levels and deleteriously contributes to atherosclerosis. Lipid binding to perlecan in bone may also modulate its flow sensory properties and the regulatory properties it conveys to osteocytes (Thompson et al., 2011; Wang, 2018).

Perlecan is localized in the periphery of stem cell niches in foetal cartilage rudiments (Melrose and Melrose, 2016) (Figures 2B–D). Perlecan regulates the attainment of stem cell pluripotency and the development of migratory chondroprogenitor stem cell lineages with roles in diarthrodial joint development, expansion of the cartilage rudiments and development of primary and secondary ossification center precursors to the cartilage growth plate cartilages. These are important features of relevance in potential repair applications that might be developed using perlecan in repair biology.

While perlecan is a component of basement membranes in vascularised tissues it also has a wide distribution in tensional and weight bearing cartilages such as the meniscus, tendon, ligament and intervertebral disc (IVD). These are predominantly avascular tissues devoid of a basement membrane, however the PCM of chondrocytes has been suggested to represent an intrinsic basement membrane around each cell (Kvist et al., 2008). Atomic force microscopy (AFM) studies demonstrate that perlecan imparts compliancy to the PCM and is cytoprotective (Guilak et al., 2021). Cell-ECM interconnections in cartilages and perlecan as a biosensor, facilitates cell-matrix communication allowing cells to perceive and respond to perturbations in their biomechanical microenvironments and to orchestrate tissue homeostasis. Perlecan also monitors the flow of cannalicular fluid in the osteocyte PCM and acts as a mechanosensor that regulates bone development (Thompson et al., 2011; Wang et al., 2014; Wijeratne et al., 2016).

Perlecan has critical roles in basement membrane in the blood brain barrier and important roles in NMJ assembly and function (Figures 5, 6). Perlecan-FGF-2 interactions in the neural stem cell niche (fractone) regulate the survival, proliferation and differentiation of neuroprogenitor stem cell populations in the sub-ventricular and dentate gyrus of the hippocampus (Kerever et al., 2007; Douet et al., 2013; Kerever et al., 2014; Mercier, 2016; Kerever and Arikawa-Hirasawa, 2021; Kerever et al., 2021; Kim et al., 2021).

The importance of perlecan to the functional weight bearing and tensile properties of cartilaginous tissues is well illustrated in SJS (Schwartz-Jampel syndrome; chondrodystrophic myotonia) (Arikawa-Hirasawa et al., 2002a) and Dyssegmental dysplasia Silverman-Handmaker type (DDSH) (Arikawa-Hirasawa et al., 2001a; Arikawa-Hirasawa et al., 2001b). The former is a relatively mild skeletal condition characterized by reduced perlecan levels in tissues; however, DDSH is a very severe condition where perlecan levels in tissues are severely depleted or totally absent. SJS is an autosomal recessive disease caused by mutation in the HSPG2 gene resulting in skeletal dysplasia and neuromuscular hyperactivity (Nicole et al., 2000; Bauché et al., 2013). In this condition mutant fibroblasts secrete reduced levels of perlecan and display impaired migratory properties but normal proliferative rates (Arikawa-Hirasawa et al., 2002a). DDSH (MIM 224410) in contrast is a very severe but extremely rare condition caused by functional null mutations in the perlecan HSPG2 gene (Arikawa-Hirasawa et al., 2001a). Less than forty DDSH cases have been reported in the literature, and only four of these were detected prenatally.

Perlecan knockout is a lethal condition. Conventional perlecan knockout (i.e., Hspg2^−/−, KO) mice die just after birth mainly due to respiratory failure (Arikawa-Hirasawa et al., 1999; Costell et al., 1999). The few mice that survive to birth display macroscopic abnormalities in cephalic development and have short squat frames with severely distorted axial and appendicular skeletal development. Furthermore, internal examination shows major abnormalities in the development of the major outflow tracts from the heart in these mice. Perlecan-null mice, form normal basement membranes but these soon deteriorate at areas of increased mechanical stress e.g., areas of myocardial contraction and brain vesicle expansion. Perlecan-null mice die around E10–12, due to heart, lung, and brain defects. Weakened embryonic heart basements membranes and “leaky” cardiomyocyte-endothelial cell interfaces result in cardiac arrest due to blood leakage into the pericardial space. Major defects in lung development also contribute to the lethality of perlecan deficiency. Abnormalities in cephalic development, distortion in normal brain laminar architecture and development of holes in the fore- and midbrain also occur. Perlecan-null mice experience severe bleeding in the lung, skin, and brain, due to weakened blood vessels. Perlecan null mice also display distorted growth plate architecture and a disturbed chondrocyte organization with a loss of normal columnar chondrocyte spatial organization and expansion and distortion of the resting, proliferative and hypertrophic zones consistent with the massive disruptions seen macroscopically in skeletal development in this mouse model. Perlecan knock-out mice are not suitable for the examination of perlecan’s roles in postnatal tissue development, but starkly demonstrate the importance of perlecan in the development of pre-natal vascular and non-vascular tissues.

In order to avoid the lethality of perlecan KO mice, conditional perlecan-deficient (Hspg2^−/−, TG) mice were developed that express the perlecan transgene only in cartilage using the Col2a1promoter and enhancer (Xu et al., 2010; Xu et al., 2016). These perlecan TG mice develop a phenotype similar to DDSH but also share features of SJS (Arikawa-Hirasawa et al., 2001a; Arikawa-Hirasawa et al., 2002a). Perlecan knockdown impacts on cartilage development and skeletogenesis and impairs the normal weight bearing properties of cartilaginous tissues and functional properties of the pericellular matrix of chondrocytes (Xu et al., 2016; Ocken et al., 2020).

In Hspg2^−/−-Tg (Hspg2^−/−; Col2a1-Hspg2^Tg/−) mice, perlecan is only expressed in cartilage but not the synovium. This results in less development of osteophytic spurs in the tibial and femoral joint margins (Arikawa-Hirasawa et al., 1999; Xu et al., 2010; Ishijima et al., 2012; Kaneko et al., 2013). A perlecan exon 3 null mouse model has also been developed. Perlecan domain-I encoded by exon 3 contains its GAG attachment points thus perlecan exon 3 null mice produce perlecan deficient in HS. A 20 kDa drop in the size of the perlecan core protein is also evident. This model has yielded important information on the role of perlecan HS in the regulation of chondrocyte behavior and in tissue homeostasis and perlecan HS mediated interactions with ECM components (Whitelock et al., 2008; Smith et al., 2010; Hayes et al., 2011a; Hayes et al., 2011b; Hayes et al., 2013; Shu et al., 2013; Hayes et al., 2014; Shu et al., 2016a; Hayes et al., 2016b; Shu et al., 2018; Shu et al., 2019; Smith and Melrose, 2019). This exon 3 null GAG free form of perlecan does not participate in growth factor and morphogen sequestration and cell signaling like the full length GAG substituted form of perlecan resulting in a loss in its ability to act as a co-receptor for the presentation of these growth factors to their cognate receptors (Zhou et al., 2004; Whitelock et al., 2008). However, there are GAG-free regions of the perlecan core protein that can also bind certain growth factors. For example, perlecan domain III can bind FGF-7 and FGF-18, however it is not known to what extent this interaction can provide the same cell proliferative and differentiative properties provided by growth factors that bind to the GAG chains of perlecan domain I. Perlecan exon 3 null HS-deficient mice do not store TGF-β1 in skin tissues like the full-length perlecan does (Shu et al., 2016a). Participation in the wound healing response, previously provided by FGF-2 and VEGF perlecan interactions and the angiogenic responses they provide, is also lost in perlecan exon 3 null mice (Zhou et al., 2004). As previously discussed, full length HS substituted perlecan has major roles in tissue and organ development and wound healing orchestrated by the binding and signaling of mitogens and morphogens with cells in a temporal and dynamic fashion. FGF-7, −18 and PDGF can also bind to perlecan domains III, IV and V, such interactions may also mediate wound healing and cell signaling responses. Binding of PDGF-BB has been mapped to domain III-2 (Kd = 8 nM), lower binding affinities are evident for domains I, IV-1 and V (Kd = 34–64 nM). PDGF-AA binds to domain III-2 (Göhring et al., 1998). Perlecan HS deficiency impairs pulmonary vascular development (Chang et al., 2015). Perlecan HS chains also recruit pericytes to pulmonary vessels. HS deficiency in perlecan attenuates hypoxia-induced pulmonary hypertension involving impaired FGF-2/FGFR1 interactions (Chang et al., 2015). Perlecan exon 3 null mice display reduced healing responses due to impaired FGF-2 and VEGF signaling (Zhou et al., 2004). The chondroprotection evident in perlecan exon 3 null mice in a post traumatic OA model may be attributable to the preservation of FGFR-3-FGF-18 signaling and perlecan domain III-mediated interactions (Shu et al., 2016b), contributing to significantly reduced joint margin osteophytosis, synovial perlecan is required for osteophyte formation in knee OA (Kaneko et al., 2013). Hspg2 exon 3 null murine chondrocytes display increased hypertrophic maturational changes and chondrocyte proliferative rates in vitro and in vivo, accelerated growth plate maturation, elevated GAG deposition, and exostosis formation in the IVD (Shu et al., 2019). Perlecan HS may thus exert repressive control over chondrocytes in mature cartilage explaining why cartilage has such a poor healing response (Garcia et al., 2021).

A model of SJS in which a 4595G to A point mutation occurs in the perlecan gene displays reduced perlecan secretion and incorporation into the PCM similar to the clinical features of human SJS (Rodgers et al., 2007; Stum et al., 2008). Mice homozygous for Hspg2C1532Y-Neo (Neo/Neo) have short stature, impaired mineralization, misshapen bones, OA-like joint dysplasias and myotonia (Rodgers et al., 2007; Stum et al., 2008).

A model of SJS has also been developed that displays congenital peripheral nerve hyper-excitability, neuromyotonia, demyelination and peripheral neuropathies (Bangratz et al., 2012). This model revealed roles for perlecan in the regulation of longitudinal elongation of myelin by Schwann cells (Echaniz-Laguna et al., 2009; Bangratz et al., 2012). Perlecan-deficient mice displaying shorter internodes, had increased levels of impaired functional voltage-gated K (+) channels (Echaniz-Laguna et al., 2009). Electrophysiological studies have demonstrated muscle fiber hyper-excitability arising from such alterations in peripheral nerve organization, muscle hypertrophy and compositional changes (Xu et al., 2010).

Cerebral artery occlusion stroke models in Yucatan miniature pigs, dogs and mice (Platt et al., 2014; Vasquez et al., 2019; Llovera et al., 2021) have facilitated examination of perlecan’s roles in the repair of the blood brain barrier following stroke. Perlecan domain V has neurogenic and neuroprotective properties and promotes angiogenic repair of the blood brain barrier (Lee et al., 2011; Bix, 2013; Marcelo and Bix, 2014). Perlecan transgenic mice have demonstrated important roles for perlecan domain V in pericyte recruitment in the promotion of BBB repair processes (Nakamura et al., 2019). IL-1 is also neuroprotective and has neuron restoring properties in experimental ischemic stroke studies (Salmeron et al., 2019). Recombinant perlecan domain V is now available for blood brain barrier repair strategies (Rnjak-Kovacina et al., 2017). Recombinant perlecan domain V decorated with HS and CS chains is a vascular PG in its own right and supports endothelial cell interactions as well as full-length perlecan (Rnjak-Kovacina et al., 2017). Repair of the blood brain barrier involves pericyte recruitment, triggered by an up-regulation in PDGFRβ (Arimura et al., 2012; Makihara et al., 2015; Shibahara et al., 2020), this drives pericyte migration required for pericyte endothelial tube repair interactions in the neurovascular unit (Hellstrom et al., 1999). Perlecan binds PDGF and promotes pericyte migration and integrin α5β1 and α2β1 mediated interactions in endothelial tube formation (Hellstrom et al., 1999; Arimura et al., 2012; Shen et al., 2012; Makihara et al., 2015; Nakamura et al., 2019).

While perlecan promotes the proliferation and differentiation of endothelial cells, as well as many other cell types, it inhibits SMC proliferation through the tumor repressor PTEN (tumor suppressor phosphatase and tensin homolog), including upregulation of FRNK (focal adhesion kinase–related non-kinase) and down regulation of FAK signalling (Walker et al., 2003; Garl et al., 2004).

HS’s interactions with FGF-2, PDGF, VEGF, HGF, BMP2, GM-CSF, angiopoietin-3, and activin A illustrate the potential of perlecan domain I as a co-receptor for growth factor delivery and receptor activation. The ITIM megakaryocyte-platelet receptor (G6b-B-R) is an additional ligand for the HS chains of perlecan domain I (Vögtle et al., 2019). G6b-B-R is highly expressed in mature megakaryocytes (MKs) that regulates platelet activation (Lord et al., 2018; Vögtle et al., 2019). Binding of G6b-B-R to perlecan HS chains mediates functional responses in MKs and platelets, negatively regulating platelet adhesion to fibrinogen and collagen. It also modulates platelet adhesion to vascular graft materials and explains the varied roles of perlecan in fibrosis (Lord et al., 2009; Lord et al., 2018).

Perlecan attached to endothelial cells in the lumen of blood vessels acts as a shear flow sensor that interacts with Ca2+ or Na+ regulating charge density at the endothelial cell surface. This also regulates membrane polarization in endothelial cells and SMCs, ion transport regulates vasoconstriction and relaxation in blood vessels (Siegel et al., 2014) and is crucial for cell-cell signalling coupled with stimulatory biophysical forces that promote cell differentiation and tissue development (Saha et al., 2018). Calcium signalling through endothelial cell TRP channels (Thakore and Earley, 2019) drives vasculogenesis (Moccia et al., 2019) and SMC contractility which in turn regulates vasodilation and blood pressure (Ishai-Michaeli et al., 1990).

While the HS chains of perlecan mediate growth factor interactions in skeletogenesis, degradation of HS in situ has been shown to improve wound healing through the re-mobilization of sequestered growth factors locally at sites of tissue repair (Ishai-Michaeli et al., 1990; Zcharia et al., 2005; Nasser, 2008). Heparanase expression in osteoblastic cells also promotes bone formation and tissue remodeling at the osteochondral interface during endochondral ossification (Kram et al., 2006).

Given the crucial role that perlecan plays in multiple biological processes, it is unsurprising that perlecan, or its components, have been utilized in multiple therapeutic applications. Perlecan’s known ability to bind, sequester, and deliver a myriad of growth factors and other bioactive molecules is a key feature that has been harnessed to translate this molecule into potential therapeutics for multiple applications, from angiogenesis to the regeneration of cartilage (Gao et al., 2021; Garcia et al., 2021). Perlecan’s ability to modulate processes in cardiovascular applications has been explored through an immune-purified form of perlecan from human coronary arterial endothelial cells. This immunopurified perlecan was used to coat expanded polytetrafluoroethylene (ePTFE) vascular grafts. Implantation of the vascular grafts into the carotid arteries of an ovine model demonstrated that the perlecan-coated grafts reduced platelet adhesion and enhanced endothelial cell growth along the implanted graft when compared with the uncoated vascular graft (Lord et al., 2009). While this study demonstrated the ability for perlecan to improve vascular graft patency, isolation of perlecan from tissues or purified from conditioned medium, can be cost-prohibitive due to the small amounts available and the quantities required.

An alternative option that has been explored is through the use of recombinant fragments of perlecan. The protein core of perlecan contains five domains, many with bioactive properties, though use of recombinant perlecan fragments has focused on the use of domain-I and -V due to these domains containing GAG attachment sites. The protein component of perlecan domain I contains three GAG attachment sites. The GAGs that decorate the perlecan domain I core protein are predominantly HS, though it may also be decorated with CS. Recombinant perlecan domain I decorated with HS has been incorporated into 3D structures or scaffolds for multiple therapeutic applications. The incorporation of perlecan domain-I into 3D structures has resulted in increased retention of FGF-2 (Yang et al., 2005), as well as BMP2 (Jha et al., 2009; Srinivasan et al., 2012; Chiu et al., 2016) for cartilage repair and regeneration. More recently, advances in fabrication and microfluidics has enabled the ability to produce growth factor gradients (Hubka et al., 2019), an approach that has been utilized to generate chemotactic gradients with FGF-2R The ability to create gradients using perlecan, perlecan fragments and other bioactive components of the ECM, has significant potential in tissue repair and regeneration, including the development of smart biomaterials and constructs. It will also greatly improve the understanding of many developmental and disease processes, e.g., in cancer biology.

As mentioned above, perlecan domain V, like domain I, contains a GAG attachment site. Recombinant perlecan has been explored in several applications due to its growth factor interactions. Recombinant perlecan domain V is substituted with HS and CS chains and promotes angiogenesis by enhancing growth factor signaling (Lin et al., 2020). When perlecan or perlecan DNA is immobilized on silk or chitosan scaffolds (Lord et al., 2017) increased vascular ingrowth and integration in vivo underlies the importance of perlecan in angiogenesis/vasculogenesis.

The complexity and nuances of perlecan’s ability to modulate biological processes has been explored by immobilization technique, and the presence of GAG chains plays a key role in the orientation of this PG (Rnjak-Kovacina et al., 2016). Immobilization of perlecan domain V by physisorption or covalently, in addition with immobilization of the protein core of perlecan domain V only, or protein core decorated with GAG chains, modulated the interaction and attachment of both endothelial cells and platelets. The ability to control the interaction of different cell types holds tremendous importance to tissue repair, for example in cardiac and vascular repair procedures where tissue grafts and implants, require cues to facilitate migration and re-endothelization of the repair tissue, whist minimizing platelet adhesion. The potential of perlecan domain V to modulate platelet adhesion has been explored by incorporating this domain onto different polymeric surfaces including poly (vinyl chloride) (Chandrasekar et al., 2021), silk (Lau et al., 2021) and chitosan (Lord et al., 2017). Incorporation of perlecan domain I plasmid DNA in conjunction with VEGF189 in a rodent wound model demonstrated increased re-epithelialization of the wound, formation of sub-endothelial tissue and neo-angiogenesis within the wound bed (Lord et al., 2017).

Peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP) have been incorporated into HA scaffolds with RGDSP and TWSKV peptide HA scaffolds significantly accelerating cell proliferation (Fowler et al., 2021). Perlecan peptide TWSKV triggers differentiation of salivary gland cells into self-assembling acini-like structures expressing salivary gland biomarkers that secrete α-amylase (Pradhan et al., 2009; Pradhan et al., 2010). Purified ECM-derived peptides have been suggested as stimulatory molecules that direct the proliferation and differentiation of progenitor cell populations. These are of potential application in tissue repair processes using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Rowland et al., 2013). Laminin-111 peptide fibrin hydrogels restore salivary gland function (Nam et al., 2017). An extensive range of laminin-111-derived peptides conjugated to chitosan scaffolds also show promise in tissue engineering applications designed to promote tissue regeneration (Hozumi et al., 2012). Laminin-111 peptide-HA hydrogels have also been shown to act as a synthetic basement membrane (Yamada et al., 2013). Conjugation of RGDSP peptide to HA gels improves cell viability, accelerates formation of epithelial spheroids, and promotes the expansion of 3D progenitor cell populations, representing the first step toward the development of an engineered salivary gland (Fowler et al., 2021). Primary salivary human progenitor stem cells undergo acinar-like differentiation in HA hydrogel cultures and incorporation of basement membrane peptides derived from perlecan and laminin-111 further directs the development of 3D salivary gland-like spheroids (Srinivasan et al., 2017). Full-length perlecan also has directive roles over the 3D development of submandibular salivary glands. Heparanase colocalizes with perlecan in submandibular gland basement membrane. Cleavage of perlecan HS side chains by heparanase regulates salivary gland branching morphogenesis by modulating FGF-10 mediated cell signaling. Heparanase releases FGF-10 from perlecan HS in the basement membrane. This increases MAPK signaling, epithelial clefting, and lateral branching thus increasing submandibular gland branching morphogenesis (Patel et al., 2007). The size and sulfation patterns of the perlecan HS side chains regulate FGF-10-mediated interactions during proliferation, salivary gland duct elongation, bud expansion, and differentiation. The spatio-temporal localization of specific HS structures in salivary tissues provides a mechanistic insight as to how salivary gland developmental processes mediated by FGF10 occur in vivo (Patel et al., 2008). FGF-10 is a multi-functional paracrine growth factor that mediates mesenchymal-epithelial signaling during tissue development, growth and disease and has significant relevance to regenerative medicine (Itoh and Ohta, 2014; Itoh, 2016).

Once damaged, articular cartilage has a notoriously poor ability to repair itself (Armiento et al., 2019). Perlecan’s important well-established roles in chondrogenesis and cartilage development (Smith et al., 2010; Melrose et al., 2012) imply potential roles in cartilage repair by recapitulating developmental roles in damaged/diseased tissues (Gao et al., 2021; Garcia et al., 2021). Perlecan promotes chondroprogenitor stem cell maturation and development of pluripotent migratory stem cell lineages with roles in diarthrodial joint formation and early cartilage development (Hayes et al., 2016a; Melrose and Melrose, 2016). Perlecan’s cartilage stabilizing properties through interactions with ECM components also establish its potential in cartilage repair (SundarRaj et al., 1995; Gomes et al., 2002; Melrose et al., 2006; Melrose et al., 2008). Perlecan domain I hydrogels have been used to establish heparin binding growth factor gradients that promote cell migration of potential use to promote cartilage repair (Hubka et al., 2019). Perlecan domain I BMP-2 hydrogels have also been shown to promote chondrogenesis and cartilage repair in a murine early OA model (Yang et al., 2006; Jha et al., 2009; Srinivasan et al., 2012). BMP-2 and BMP-9 can also promote chondrogenic differentiation of human multipotential mesenchymal stem cells (Majumdar et al., 2001; Shestovskaya et al., 2021). Perlecan domain I collagen I fibril scaffolds have been used as an FGF-2 delivery system that could also be useful in cartilage repair strategies (Yang et al., 2005). FGF-2 directs non-committed mesenchymal stem cells to a chondrogenic phenotype (Shu et al., 2016c).

The duration of synaptic transmission of impulses at the NMJ is chiefly controlled by acetylcholinesterase (AChE), an enzyme which hydrolyzes acetylcholine (ACh) to choline and acetate (Katz and Miledi, 1973; Rosenberry, 1979). Perlecan, nidogens, glycoproteins and collagen fill the synaptic cleft and are relevant for signal transduction as well as the maintenance of mechanical strength in the NMJ. At the NMJ, the most abundant form of AChE is the AChE_T which is anchored to the synaptic basal lamina by non-fibrillar collagen Q (ColQ) (Krejci et al., 1991; Krejci et al., 1997). ColQ, a triple helical structure, binds to perlecan domain 1 via two heparan sulfate PG binding motifs and to muscle specific kinase (MuSK) respectively (Rotundo et al., 1997; Casanueva et al., 1998; Deprez et al., 2003; Cartaud et al., 2004; Nakata et al., 2013).

Experimental reports demonstrate the functional relationship between perlecan and AChE (Peng et al., 1999; Arikawa-Hirasawa et al., 2002b; Massoulié and Millard, 2009; Maselli et al., 2012) and the cellular co-localization of perlecan and AChE at the synaptic basal lamina in vitro and in pre-clinical models of the NMJ (Peng et al., 1999; Arikawa-Hirasawa et al., 2002b; Massoulié and Millard, 2009; Maselli et al., 2012). Perlecan null mice lack AChE clusters at the NMJ (Peng et al., 1999; Martinez-Pena y Valenzuela et al., 2007).

Ischemic stroke is a leading cause of permanent disability and mortality and there is an urgent need for adjunctive therapies for concurrent administration with existing clot-busting (tissue plasminogen activator, tPA) and endovascular clot removal (thrombectomy) treatments. Whilst these primary treatments remove the underlying cause of the stroke, they do little to reverse or prevent subsequent brain injury. Perlecan domain V is potentially one such therapy whose efficacy has been experimentally demonstrated in both young and aged rodent models of ischemic stroke, where it has been reported to have neuroprotective, neuroreparative and functionally restorative effects (Lee et al., 2011; Bix et al., 2013; Trout et al., 2021). These benefits of perlecan domain V have been linked to its maintenance of the BBB through α5β1 integrin modulation and increased VEGF secretion (Clarke et al., 2012; Nakamura et al., 2019). Bix and colleagues showed that brain expression of perlecan is substantially and chronically increased following experimental and clinical ischemic stroke (Lee et al., 2011; Bix et al., 2013; Trout et al., 2021). This observation has led to the hypothesis that perlecan plays a significant role in brain repair following ischemic insult. Accordingly, larger infarct volumes and severe motor-sensory deficits in transgenic mice with only 10% of total perlecan bioavailability (perlecan hypomorphs), when compared to age-matched wild type controls, have been independently reported and corroborated (Lee et al., 2011; Nakamura et al., 2019). This suggests that physiologic levels of perlecan are important for the brain to repair itself after stroke, and that diminished perlecan levels are detrimental for stroke outcomes. It follows that exogenously administered perlecan domain V would enhance the brain’s ability in self-repair by increasing the availability of perlecan for BBB maintenance, thereby improving brain injury and functional deficits following ischemic stroke.

Perlecan promotes blood brain barrier repair following acute brain injury by modulating the survival and migration of PDGFRβ-positive pericytes to the ischemic brain region (Arimura et al., 2012; Shen et al., 2012; Nakamura et al., 2016; Hoffmann et al., 2018; Nakamura et al., 2019). Perlecan domain V binds to PDGFRβ and α5β1 in order to maintain blood brain integrity in the injured brain area (Nakamura et al., 2019) Also, following ischemic brain injury, α5β1 integrin expression increases and the exogenously administered perlecan domain V binds to this integrin on endothelial cells to bring about the increased secretion of VEGF (Clarke et al., 2012). The elevated levels of VEGF enables angiogenesis via the downstream activation of PI3K-Akt, MEK-ERK and HIF-1α (Milner et al., 2008; Clarke et al., 2012). Surprisingly, the increased VEGF does not worsen the BBB disruption following perlecan domain V administration after ischemic stroke (Lee et al., 2011). The intraperitoneal administration of perlecan domain V decreases infarct volume as well as immuno-expression of markers of apoptosis (TUNEL and Caspase 3 cleavage) (Lee et al., 2011). More so, perlecan domain V modulates the activation and proliferation of astrocytes and ameliorates astrogliosis in vitro and in vivo. This indicates that perlecan domain V rescues neuronal death and may actively dampen scar formation after ischemic stroke suggesting it has instructive regulatory properties in tissue fibrosis (Lord et al., 2018). Matricryptin peptides derived from ECM components also have roles in tissue remodeling (Amruta et al., 2020; de Castro Brás and Frangogiannis, 2020). The in vivo benefits of perlecan domain V have been evaluated in rodents using various models of ischemic using middle cerebral artery occlusion which includes tandem ipsilateral common carotid and middle cerebral arteries occlusion (CCA/MCAO) model that induces mild cortical lesion (Trout et al., 2021), endothelin (ET)-1 injection (mild to moderate brain lesion) (Lee et al., 2011), photothrombotic (Bix et al., 2013) and intraluminal filament MCAO model -severe brain injury.

The therapeutic potential of exogenously administered perlecan domain V in a severe pre-clinical ischemic stroke model (photothrombotic and intraluminal MCAO) is essentially dependent on how quickly the first dose of perlecan domain V is administered after ischemic stroke induction. A recent report by Nakamura et al. (Nakamura et al., 2019) found mild effects of exogenously administered recombinant perlecan domain V (5 mg/kg) on infarct volume after intraluminal MCAO induction. Perlecan domain V was administered 24 h after reperfusion and daily for three to four consecutive days. However, Bix and colleagues had previously reported that the timely administration of perlecan domain V (2 mg/kg) after severe ischemic stroke (photothrombotic model) is vital for neuroprotective benefits (Clarke et al., 2012). Indeed, the specific report indicates that when perlecan domain V is administered 3 hours after stroke induction, infarct volume is smaller when compared to perlecan domain V administration when commenced six, twelve and 24 hours post-stroke induction. Importantly, infarct volume increases as the start of perlecan domain V treatment after stroke induction period increases. Additionally, recent findings show a significant decrease in infarct volume when perlecan domain V was co-administered during reperfusion in a mouse model of intraluminal MCAO (Biose et al., 2021).

Early reports have shown that human recombinant LG3, the C-terminal-most portion of perlecan domain V, may confer the neuroprotective efficacy of full length perlecan domain V (Saini and Bix, 2012; Bix, 2013). Like domain V, LG3 is increased in the compromised brain hemisphere after 24 hours of transient MCAO and it persistently remains elevated for 3 days after stroke in rats.³ Similarly, 2 hours of oxygen-glucose deprivation (OGD) in astrocytes and neuron cultures substantially increases LG3 levels. LG3 is anti-apoptotic as it decreases Caspase 3 after OGD-reperfusion (Lee et al., 2011; Saini and Bix, 2012). Also, LG3 preserved the integrity of fetal cortical neurons by reducing Caspase 3 immunohistochemistry after in vitro OGD when compared to control dishes (Saini and Bix, 2012). Investigations are ongoing to determine the effect of LG3 on infarct volume and motor-sensory functions in various preclinical models of ischemic stroke (Biose et al., 2021).

In summary, perlecan is actively processed for brain repair after ischemic stroke and is neuroprotective, improves angiogenesis and motor-sensory functions and modulates astrocyte activity when administered in a timely manner following stroke. Furthermore, LG3 which confers the biological activity of perlecan domain V in infarct volume reduction may have potential adjunctive stroke therapeutic value in preventing mortality among clinical stroke patients (Biose et al., 2021).