Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

Biocatalytic Buoyancy-Driven Nanobots for Autonomous Cell Recognition and Enrichment.

Autonomously self-propelled nanoswimmers represent the next-generation nano-devices for bio- and environmental technology. However, current nanoswimmers generate limited energy output and can only move in short distances and duration, thus are struggling to be applied in practical challenges, such as living cell transportation. Here, we describe the construction of biodegradable metal-organic framework based nanobots with chemically driven buoyancy to achieve highly efficient, long-distance, directional vertical motion to "find-and-fetch" target cells. Nanobots surface-functionalized with antibodies against the cell surface marker carcinoembryonic antigen are exploited to impart the nanobots with specific cell targeting capacity to recognize and separate cancer cells. We demonstrate that the self-propelled motility of the nanobots can sufficiently transport the recognized cells autonomously, and the separated cells can be easily collected with a customized glass column, and finally regain their full metabolic potential after the separation. The utilization of nanobots with easy synthetic pathway shows considerable promise in cell recognition, separation, and enrichment.

Engineered short forms of perlecan enhance angiogenesis by potentiating growth factor signalling.

Growth factors are key molecules involved in angiogenesis, a process critical for tissue repair and regeneration. Despite the potential of growth factor delivery to stimulate angiogenesis, limited clinical success has been achieved with this approach. Growth factors interact with the extracellular matrix (ECM), and particularly heparan sulphate (HS), to bind and potentiate their signalling. Here we show that engineered short forms of perlecan, the major HS proteoglycan of the vascular ECM, bind and signal angiogenic growth factors, including fibroblast growth factor 2 and vascular endothelial growth factor-A. We also show that engineered short forms of perlecan delivered in porous chitosan biomaterial scaffolds promote angiogenesis in a rat full thickness dermal wound model, with the fusion of perlecan domains I and V leading to superior vascularisation compared to native endothelial perlecan or chitosan scaffolds alone. Together, this study demonstrates the potential of engineered short forms of perlecan delivered in chitosan scaffolds as next generation angiogenic therapies which exert biological activity via the potentiation of growth factors.

Comparison of protein quantification methods for protein encapsulation with ZIF-8 metal-organic frameworks.

The use of metal-organic frameworks (MOFs) as delivery systems for biologically functional macromolecules has been explored widely in recent years due to their ability to protect their payload from a wide range of harsh conditions. Given the wide usage and diversity of potential applications, optimising the encapsulation efficiency by MOFs for different biological is of particular importance. Here, several protein quantitation methods and report were compared on the accuracy, practicality, limitations, and sensitivity of these methods to assess the encapsulation efficiency of zeolitic imidazolate frameworks (ZIF)-8 MOFs for two common biologicals commonly used in nanomedicine, bovine serum albumin (BSA), and the enzyme catalase (CAT). Using these methods, ZIF-8 encapsulation of BSA and CAT was confirmed to enrich for high molecular weight and glycosylated protein forms. However, contrary to most reports, a high degree of variance was observed across all methods assessed, with fluorometric quantitation providing the most consistent results with the lowest background and greatest dynamic range. While bicinchoninic acid (BCA) assay has showed greater detection range than the Bradford (Coomassie) assay, BCA and Bradford assays were found to be susceptible to background from the organic "MOF" linker 2-methylimidazole, reducing their overall sensitivity. Finally, while very sensitive and useful for assessing protein quality SDS-PAGE is also susceptible to confounding artifacts and background. Given the increasing use of enzyme delivery using MOFs, and the diversity of potential uses in biomedicine, identifying a rapid and efficient method of assessing biomolecule encapsulation is key to their wider acceptance.

Recombinant perlecan domain V covalently immobilized on silk biomaterials via plasma immersion ion implantation supports the formation of functional endothelium.

Strategies to promote rapid formation of functional endothelium are required to maintain blood fluidity and regulate smooth muscle cell proliferation in synthetic vascular conduits. In this work, we explored the biofunctionalization of silk biomaterials with recombinantly expressed domain V of human perlecan (rDV) to promote endothelial cell interactions and the formation of functional endothelium. Perlecan is essential in vascular development and homeostasis and rDV has been shown to uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions, both key contributors of vascular graft failure. rDV was covalently immobilized on silk using plasma immersion ion implantation (PIII), a simple one-step surface treatment process which enables strong immobilization in the absence of chemical cross-linkers. rDV immobilization on surface-modified silk was assessed for amount, orientation, and bio-functionality in terms of endothelial cell interactions and functional endothelial layer formation. rDV immobilized on PIII-treated silk (rDV-PIII-silk) supported rapid endothelial cell adhesion, spreading, and proliferation to form functional endothelium, as evidenced by the expression of vinculin and VE-cadherin markers. Taken together, the results provide evidence for the potential of rDV-PIII-silk as a biomimetic vascular graft material.

A Biomolecular Toolbox for Precision Nanomotors.

The application of nanomotors for cancer diagnosis and therapy is a new and exciting area of research, which when combined with precision nanomedicine, promises to solve many of the issues encountered by previous development of passive nanoparticles. The goal of this article is to introduce nanomotor and nanomedicine researchers to the deep pool of knowledge available regarding cancer cell biology and biochemistry, as well as provide a greater appreciation of the complexity of cell membrane compositions, extracellular surfaces, and their functional consequences. A short description of the nanomotor state-of-art for cancer therapy and diagnosis is first provided, as well as recommendations for future directions of the field. Then, a biomolecular targeting toolbox has been collated for researchers looking to apply their nanomaterial of choice to a biological setting, as well as providing a glimpse into currently available clinical therapies and technologies. This toolbox contains an overview of different classes of targeting molecules available for high affinity and specific targeting and cell surface targets to aid researchers in the selection of a clinical disease model and targeting methodology. It is hoped that this review will provide biological context, inspiration, and direction to future nanomotor and nanomedicine research.

Tuning the intentional corona of cerium oxide nanoparticles to promote angiogenesis via fibroblast growth factor 2 signalling.

Inadequate angiogenesis is a hallmark of conditions including cardiovascular diseases, stroke and chronic diabetic wounds, which exhibit tissue ischaemia ensuring that therapeutic strategies to promote angiogenesis are of great interest. However, many angiogenic treatments involve the delivery of growth factors which have limited clinical success due to poor stability, high manufacturing cost and poor efficacy. Cerium oxide nanoparticles (nanoceria) can either promote or inhibit angiogenesis depending on their surface corona chemistry. Here, nanoceria were functionalized with an intentional heparin corona, a polysaccharide which binds and signals growth factors, of different chain lengths and surface grafting density to establish their effect on angiogenesis. These nanoparticles promoted angiogenesis in vivo with the surface grafting density positively correlated with angiogenesis over the widest concentration range; however, chain length did not play a role. The heparin-nanoceria supported fibroblast growth factor 2 (FGF2) signalling in vitro and promoted FGF2-mediated angiogenesis in vivo. The nanoparticles were internalized by endothelial cells in vitro where they trafficked to the lysosomes and reduced cell viability suggesting that the angiogenic activity of heparin-nanoceria is mediated in the extracellular environment. Together, this study adds to our knowledge of the angiogenic effects of heparin-nanoceria towards finding new angiogenic treatments.

Chondroitin Sulfate Proteoglycan 4 as a Marker for Aggressive Squamous Cell Carcinoma.

Chondroitin sulfate (CS) proteoglycan 4 (CSPG4) is a cell surface proteoglycan that is currently under investigation as a marker of cancer malignancy, and as a potential target of anticancer drug treatment. CSPG4 acts as a driver of tumourigenesis by regulating turnover of the extracellular matrix (ECM) to promote tumour cell invasion, migration as well as inflammation and angiogenesis. While CSPG4 has been widely studied in certain malignancies, such as melanoma, evidence is emerging from global gene expression studies, which suggests a role for CSPG4 in squamous cell carcinoma (SCC). While relatively treatable, lack of widely agreed upon diagnostic markers for SCCs is problematic, especially for clinicians managing certain patients, including those who are aged or infirm, as well as those with underlying conditions such as epidermolysis bullosa (EB), for which a delayed diagnosis is likely lethal. In this review, we have discussed the structure of CSPG4, and quantitatively analysed CSPG4 expression in the tissues and pathologies where it has been identified to determine the usefulness of CSPG4 expression as a diagnostic marker and therapeutic target in management of malignant SCC.

Surface Biofunctionalization of Silk Biomaterials Using Dityrosine Cross-Linking.

Biofunctionalization of silk biomaterial surfaces with extracellular matrix (ECM) molecules, cell binding peptides, or growth factors is important in a range of applications, including tissue engineering and development of implantable medical devices. Passive adsorption is the most common way to immobilize molecules of interest on preformed silk biomaterials but can lead to random molecular orientations and displacement from the surface, limiting their applications. Herein, we developed techniques for covalent immobilization of biomolecules using enzyme- or photoinitiated formation of dityrosine bonds between the molecule of interest and silk. Using recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) as a model molecule, we demonstrated that rDV can be covalently immobilized via dityrosine cross-linking without the need to modify rDV or silk biomaterials. Dityrosine-based immobilization resulted in a different molecular orientation to passively absorbed rDV with less C- and N-terminal region exposure on the surface. Dityrosine-based immobilization supported functional rDV immobilization where immobilized rDV supported endothelial cell adhesion, spreading, migration, and proliferation. These results demonstrate the utility of dityrosine-based cross-linking in covalent immobilization of tyrosine-containing molecules on silk biomaterials in the absence of chemical modification, adding a simple and accessible technique to the silk biofunctionalization toolbox.

Regulation of FGF-2, FGF-18 and Transcription Factor Activity by Perlecan in the Maturational Development of Transitional Rudiment and Growth Plate Cartilages and in the Maintenance of Permanent Cartilage Homeostasis.

The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environments. Nuclear perlecan may modulate chromatin structure and transcription factor access to DNA and gene regulation. Snail-1, a mesenchymal marker and transcription factor, signals through FGFR-3 to promote chondrogenesis and maintain Acan and type II collagen levels in articular cartilage, but prevents further tissue expansion. Pre-hypertrophic growth plate chondrocytes also express high Snail-1 levels, leading to cessation of Acan and CoI2A1 synthesis and appearance of type X collagen. Perlecan differentially regulates FGF-2 and FGF-18 to maintain articular cartilage homeostasis, rudiment and growth plate cartilage growth, and maturational changes including mineralization, contributing to skeletal growth.

ROS-Mediated Anti-Angiogenic Activity of Cerium Oxide Nanoparticles in Melanoma Cells.

Angiogenesis plays a key role in cancer progression, including transition to the metastatic phase via reactive oxygen species (ROS)-dependent pathways, among others. Antivascular endothelial growth factor (VEGF) antibodies have been trialed as an anti-angiogenic therapy for cancer but are associated with high cost, limited efficacy, and side effects. Cerium oxide nanoparticles (nanoceria) are promising nanomaterials for biomedical applications due to their ability to modulate intracellular ROS. Nanoceria can be produced by a range of synthesis methods, with chemical precipitation as the most widely explored. It has been reported that chemical precipitation can fine-tune primary particle size where a limited number of synthesis parameters were varied. Here, we explore the effect of temperature, precipitating agent concentration and rate of addition, stirring rate, and surfactant concentration on nanoceria primary particle size using a fractional factorial experimental design approach. We establish a robust synthesis method for faceted nanoceria with primary particle diameters of 5-6 nm. The nanoceria are not cytotoxic to a human melanoma cell line (Mel1007) at doses up to 400 µg/mL and are dose-dependently internalized by the cells. The intracellular ROS level for some cells that internalized the nanoceria is reduced, which correlates with a dose-dependent reduction in angiogenic gene expression including VEGF. These findings contribute to our knowledge of the anti-angiogenic effects of nanoceria and help to develop our understanding of potentially new anti-angiogenic agents for combination cancer therapies.

Effect of Recombinant Human Perlecan Domain V Tethering Method on Protein Orientation and Blood Contacting Activity on Polyvinyl Chloride.

Surface modification of biomaterials is a promising approach to control biofunctionality while retaining the bulk biomaterial properties. Perlecan is the major proteoglycan in the vascular basement membrane that supports low levels of platelet adhesion but not activation. Thus, perlecan is a promising bioactive for blood-contacting applications. This study furthers the mechanistic understanding of platelet interactions with perlecan by establishing that platelets utilize domains III and V of the core protein for adhesion. Polyvinyl chloride (PVC) is functionalized with recombinant human perlecan domain V (rDV) to explore the effect of the tethering method on proteoglycan orientation and bioactivity. Tethering of rDV to PVC is achieved via either physisorption or covalent attachment via plasma immersion ion implantation (PIII) treatment. Both methods of rDV tethering reduce platelet adhesion and activation compared to the pristine PVC, however, the mechanisms are unique for each tethering method. Physisorption of rDV on PVC orientates the molecule to hinder access to the integrin-binding region, which inhibits platelet adhesion. In contrast, PIII treatment orientates rDV to allow access to the integrin-binding region, which is rendered antiadhesive to platelets via the glycosaminoglycan (GAG) chain. These effects demonstrate the potential of rDV biofunctionalization to modulate platelet interactions for blood contacting applications.

Macrophages bind LDL using heparan sulfate and the perlecan protein core.

The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.

Biomimetic silk biomaterials: Perlecan-functionalized silk fibroin for use in blood-contacting devices.

Blood compatible materials are required for the development of therapeutic and diagnostic blood contacting devices as blood-material interactions are a key factor dictating device functionality. In this work, we explored biofunctionalization of silk biomaterials with a recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) towards the development of blood compatible surfaces. Perlecan and rDV are of interest in vascular device development as they uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions. rDV was covalently immobilized on silk biomaterials using plasma immersion ion implantation (PIII), a new method of immobilizing proteins on silk biomaterials that does not rely on modification of specific amino acids in the silk protein chain, and compared to physisorbed and carbodiimide immobilized rDV. Untreated and treated silk biomaterials were examined for interactions with blood components with varying degrees of complexity, including isolated platelets, platelet rich plasma, blood plasma, and whole blood, both under agitated and flow conditions. rDV-biofunctionalized silk biomaterials were shown to be blood compatible in terms of platelet and whole blood interactions and the PIII treatment was shown to be an effective and efficient means of covalently immobilizing rDV in its bioactive form. These biomimetic silk biomaterials are a promising platform toward development of silk-based blood-contacting devices for therapeutic, diagnostic, and research applications. STATEMENT OF SIGNIFICANCE: Blood compatible materials are required for the development of therapeutic and diagnostic blood contacting devices as blood-material interactions are a key factor dictating device functionality. In this work, we explored biofunctionalization of silk biomaterials with a recombinantly expressed domain V (rDV) of the human basement membrane proteoglycan perlecan towards the development of blood compatible surfaces. Perlecan and rDV are of interest in vascular device development as they uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions. rDV was covalently immobilized on silk biomaterials using plasma immersion ion implantation (PIII), a new method of immobilizing proteins on silk biomaterials that does not rely on modification of specific amino acids in the silk protein chain. These biomimetic silk biomaterials are a promising platform toward development of silk-based blood-contacting devices for therapeutic, diagnostic, and research applications.

A Biomimetic Approach toward Enhancing Angiogenesis: Recombinantly Expressed Domain V of Human Perlecan Is a Bioactive Molecule That Promotes Angiogenesis and Vascularization of Implanted Biomaterials.

Angiogenic therapy involving delivery of pro-angiogenic growth factors to stimulate new blood vessel formation in ischemic disease is promising but has seen limited clinical success due to issues associated with the need to deliver supra-physiological growth factor concentrations. Bio-inspired growth factor delivery utilizing the native growth factor signaling roles of the extracellular matrix proteoglycans has the potential to overcome many of the drawbacks of angiogenic therapy. In this study, the potential of the recombinantly expressed domain V (rDV) of human perlecan is investigated as a means of promoting growth factor signaling toward enhanced angiogenesis and vascularization of implanted biomaterials. rDV is found to promote angiogenesis in established in vitro and in vivo angiogenesis assays by potentiating endogenous growth factor signaling via its glycosaminoglycan chains. Further, rDV is found to potentiate fibroblast growth factor 2 (FGF2) signaling at low concentrations that in the absence of rDV are not biologically active. Finally, rDV immobilized on 3D porous silk fibroin biomaterials promotes enhanced vascular ingrowth and integration of the implanted scaffolds with the surrounding tissue. Together, these studies demonstrate the important role of this biologically active perlecan fragment and its potential in the treatment of ischemia in both native and bioengineered tissues.

The Inter-α-Trypsin Inhibitor Family: Versatile Molecules in Biology and Pathology.

Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children.

Microchannels Are an Architectural Cue That Promotes Integration and Vascularization of Silk Biomaterials in Vivo.

Functional integration of implanted biomaterials and bioengineered tissues in vivo requires effective and timely vascular ingrowth. While many vascularization strategies rely on delivery of angiogenic growth factors or endothelial cells to promote vascular ingrowth, the effect of physical and architectural features of biomaterials on the vascularization process is less well understood. Microchannels are a simple, accessible architectural feature frequently engineered into 3D biomaterials to promote mass transfer. In this study, the effect of microchannels on the integration and vascularization of 3D porous silk scaffolds was explored over a 14 week period. An array of 508 µm diameter microchannels spanning the length of critically sized, porous silk scaffolds significantly improved tissue ingrowth into the constructs. At week 6, all silk scaffolds (n = 8) with microchannels showed complete tissue infiltration throughout the construct, while only one of eight (12.5%) did so in the absence of microchannels. The presence of microchannels improved silk scaffold vascularization with significantly more vessels per unit area in the presence of microchannels. The vessel size distribution was similar in both scaffold types, but a shift in distribution toward smaller vessels was observed in the presence of microchannels. The blood vessels in silk scaffolds were perfused, functional and connected to the animal's cardiovascular system, as demonstrated by the presence of red blood cells in the vessel lumens, and effective delivery of a contrast agent the vessels inside the scaffold. This study demonstrates the utility of microchannels as a simple architectural feature that significantly improves vascularization and integration of implanted biomaterials.

CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan.

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.