Perlecan Deficiency Research Articles

Increased Risk of Aortic Dissection with Perlecan Deficiency.

Perlecan (HSPG2), a basement membrane-type heparan sulfate proteoglycan, has been implicated in the development of aortic tissue. However, its role in the development and maintenance of the aortic wall remains unknown. Perlecan-deficient mice (Hspg2−/−-Tg: Perl KO) have been found to show a high frequency (15–35%) of aortic dissection (AD). Herein, an analysis of the aortic wall of Perl KO mice revealed that perlecan deficiency caused thinner and partially torn elastic lamina. Compared to the control aortic tissue, perlecan-deficient aortic tissue showed a significant decrease in desmosine content and an increase in soluble tropoelastin levels, implying the presence of immature elastic fibers in Perl KO mice. Furthermore, the reduced expression of the smooth muscle cell contractile proteins actin and myosin in perlecan-deficient aortic tissue may explain the risk of AD. This study showed that a deficiency in perlecan, which is localized along the elastic lamina and at the interface between elastin and fibrillin-1, increased the risk of AD, largely due to the immaturity of extracellular matrix in the aortic tissue. Overall, we proposed a new model of AD that considers the deficiency of extracellular molecule perlecan as a risk factor.

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

Trabecular Bone Deficit and Enhanced Anabolic Response to Re-Ambulation after Disuse in Perlecan-Deficient Skeleton.

Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.

Perlecan/Hspg2 deficiency impairs bone’s calcium signaling and associated transcriptome in response to mechanical loading

Perlecan, a heparan sulfate proteoglycan, acts as a mechanical sensor for bone to detect external loading. Deficiency of perlecan increases the risk of osteoporosis in patients with Schwartz-Jampel Syndrome (SJS) and attenuates loading-induced bone formation in perlecan deficient mice (Hypo). Considering that intracellular calcium [Ca2+]i is an ubiquitous messenger controlling numerous cellular processes including mechanotransduction, we hypothesized that perlecan deficiency impairs bone's calcium signaling in response to loading. To test this, we performed real-time [Ca2+]i imaging on in situ osteocytes of adult murine tibiae under cyclic loading (8N). Relative to wild type (WT), Hypo osteocytes showed decreases in the overall [Ca2+]i response rate (-58%), calcium peaks (-33%), cells with multiple peaks (-53%), peak magnitude (-6.8%), and recovery speed to baseline (-23%). RNA sequencing and pathway analysis of tibiae from mice subjected to one or seven days of unilateral loading demonstrated that perlecan deficiency significantly suppressed the calcium signaling, ECM-receptor interaction, and focal adhesion pathways following repetitive loading. Defects in the endoplasmic reticulum (ER) calcium cycling regulators such as Ryr1/ryanodine receptors and Atp2a1/Serca1 calcium pumps were identified in Hypo bones. Taken together, impaired calcium signaling may contribute to bone's reduced anabolic response to loading, underlying the osteoporosis risk for the SJS patients.

Abstract TP276: Perlecan Is Required for the Maintenance of the Blood-Brain Barrier through the Interaction with Pericytes in a Mouse Ischemic Stroke Model

Introduction: The disruption of the blood-brain barrier (BBB) is a contributing factor for the deterioration of brain damages in ischemic stroke. Recent studies revealed that microvascular pericytes are involved in the maintenance of the BBB and in the repair process through platelet-derived growth factor receptor β (PDGFRβ), the expression of which is increased around the ischemic lesion. Here we focus on perlecan, the major heparan sulfate proteoglycan in the basement membrane (BM). It is expressed by endothelial cells in the brain and is implicated in many biological functions. In this report, we have studied the role of Perlecan in the BBB breakdown and in the subsequent repair process after ischemic stroke in a mouse model. We hypothesized that perlecan may play a protective role in the disruption of BBB through the interaction with pericytes after ischemic stroke. Methods: To elucidate the role of perlecan in the brain vasculature, we induced a 60-minute transient middle cerebral artery occlusion (MCAO) in adult conditional perlecan -deficient ( Perlecan -/- -Tg) mice, which express the perlecan transgene only in the cartilage to rescue the perinatal lethality of perlecan -deficient mice. Results: Although the BBB formation and function in the brain vasculature appeared to be unaffected in Perlecan -/- -Tg mice under healthy condition, Perlecan -/- -Tg mice demonstrated larger infarct volumes and more BBB leakage than control mice on post-surgery day (PSD) 2 after MCAO. Perlecan -/- -Tg mice exhibited less PDGFRβ-positive pericytes around the ischemic lesion on PSD 3 to 7 than control mice, suggesting that the perlecan deficiency suppressed pericyte activation. At a mechanistic level, integrin α5, a potential receptor for perlecan, was detectable in both endothelial cells and pericytes in the ischemic lesion, suggesting that endothelial cell-derived perlecan may regulate pericyte activation in response to ischemia through integrin α5. Conclusions: Our results suggest that perlecan is required for the activation of pericytes and thereby, contributing to the endothelial cell-pericyte integrity in the BBB maintenance after ischemic stroke.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle

The autophagy–lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

The dependences of osteocyte network on bone compartment, age, and disease.

Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the “nodes” in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the “bandwidth” of individual links in a computer network). Age, in the range of our study (15–32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.

Perlecan deficiency causes endothelial dysfunction by reducing the expression of endothelial nitric oxide synthase.

Perlecan is a major heparan sulfate proteoglycan found in the subendothelial extracellular matrix of the vascular wall. The aim of this study was to investigate the role of perlecan in the regulation of vascular tone. A previously developed conditional perlecan‐deficient mouse model was used to measure changes in the isometric force of isolated aortic rings. The vessels were first precontracted with phenylephrine, and then treated with increasing concentrations of vasorelaxants. Endothelium‐dependent relaxation, elicited by acetylcholine, was significantly reduced in the perlecan‐deficient aortas, whereas endothelium‐independent relaxation caused by the exogenous nitric oxide donor sodium nitroprusside remained well preserved. The expression of the endothelial nitric oxide synthase (eNOS) gene, detected by real‐time polymerase chain reaction, was significantly decreased in the perlecan‐deficient aortas. The expression of eNOS protein detected using Western blotting was also significantly decreased in the perlecan‐deficient aortas. We examined the role of perlecan in eNOS gene expression by creating perlecan knockdown human aortic endothelial cells using small interfering RNA (siRNA) for perlecan. Perlecan gene expression was significantly reduced in the perlecan siRNA‐treated cells, resulting in a significant decrease in eNOS gene expression. Perlecan deficiency induced endothelial dysfunction, as indicated by a reduction in endothelium‐dependent relaxation due, at least partly, to a reduction in eNOS expression. These findings suggest that perlecan plays a role in the activation of eNOS gene expression during normal growth processes.

Perlecan is required for FGF-2 signaling in the neural stem cell niche

In the adult subventricular zone (neurogenic niche), neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

Perlecan-Containing Pericellular Matrix Regulates Solute Transport and Mechanosensing Within the Osteocyte Lacunar-Canalicular System

The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both "outside-in" (mechanosensing) and "inside-out" (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (∼100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (-35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.

Perlecan-Deficient Mutation Impairs Corneal Epithelial Structure

To elucidate the role of perlecan (Hspg2), a large multidomain heparan sulfate proteoglycan expressed in the basement membrane, in the structure of the corneal epithelium. A previously developed perlecan-deficient (Hspg2⁻/⁻-Tg) mouse model was used. Histologic analysis of their corneas was performed by light and transmission electron microscopy. The localization of perlecan in the corneas of wild-type (WT) mice and Hspg2⁻/⁻-Tg mice was examined by immunohistochemistry. The effects of perlecan deficiency on corneal epithelial structure was analyzed with respect to the expression of corneal epithelial proliferation and differentiation markers, such as Ki67, cytokeratin12 (K12), connexin43 (Cx43), Notch1, and Pax6 by immunohistochemistry and real-time polymerase chain reaction (PCR). The Hspg2⁻/⁻-Tg mice had microphthalmos and a thinner corneal epithelium compared with that of the WT mice. Perlecan was localized in the corneal epithelial basement membrane in the WT mice, but not in the Hspg2⁻/⁻-Tg mice. The Hspg2⁻/⁻-Tg corneal epithelium exhibited thinner wing cell layers and a decreased number of Ki67-positive cells, but no dead cells, compared with the WT corneal epithelium. Immunohistochemistry and real-time PCR analysis revealed a significantly decreased expression of corneal epithelial differentiation markers such as K12, Cx43, Notch1, and Pax6 in Hspg2⁻/⁻-Tg mice, compared with those of the WT mice. The findings of this study highlight a strong correlation between the presence of perlecan in the basement membrane and the structure of corneal epithelium and that the perlecan-deficient mutation impairs corneal epithelial structure.

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2−/−) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2−/− growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2−/− growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2−/− growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2−/− mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.

Perlecan deficiency causes muscle hypertrophy, a decrease in myostatin expression, and changes in muscle fiber composition

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2−/−-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2−/−-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2−/−-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2−/−-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2−/−-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2−/−-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2−/−-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2−/−-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2−/−-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2−/−-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.

P27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

Electrophysiological studies in a mouse model of Schwartz–Jampel syndrome demonstrate muscle fiber hyperactivity of peripheral nerve origin

Schwartz-Jampel syndrome (SJS) is an autosomal-recessive condition characterized by muscle stiffness and chondrodysplasia. It is due to loss-of-function hypomorphic mutations in the HSPG2 gene that encodes for perlecan, a proteoglycan secreted into the basement membrane. The origin of muscle stiffness in SJS is debated. To resolve this issue, we performed an electrophysiological investigation of an SJS mouse model with a missense mutation in the HSPG2 gene. Compound muscle action potential amplitudes, distal motor latencies, repetitive nerve stimulation tests, and sensory nerve conduction velocities of SJS mice were normal. On electromyography (EMG), neuromyotonic discharges, that is, bursts of motor unit action potentials firing at high rates (120-300 HZ), were constantly observed in SJS mice in all muscles, except in the diaphragm. Neuromyotonic discharges were not influenced by general anesthesia and disappeared with curare administration. They persisted after complete motor nerve section, terminating only with Wallerian degeneration. These results demonstrate that perlecan deficiency in SJS provokes a neuromyotonic syndrome. The findings further suggest a distal axonal localization of the generator of neuromyotonic discharges. SJS should now be considered as an inherited disorder with peripheral nerve hyperexcitability.

Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz–Jampel syndrome neuromyotonia

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

Targeting Perlecan in Human Keratinocytes Reveals Novel Roles for Perlecan in Epidermal Formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.