Treatment with transducible phosphopeptide analogues of the small heat shock-related protein, HSP20, after experimental subarachnoid hemorrhage: prevention and reversal of delayed decreases in cerebral perfusion.

OBJECT: Delayed vasospasm is a significant cause of morbidity and mortality after subarachnoid hemorrhage (SAH). Proteomic therapeutics offers a new modality in which biologically active proteins or peptides are transduced into cells via covalent linkage to cell permeant peptides (CPPs). The hypothesis of this study was that either intrathecal or intravenous delivery of a phosphopeptide mimetic of the small heat shock-related protein, HSP20, linked to a CPP, would inhibit delayed decreases in cerebral perfusion after experimental SAH in a rat model. METHODS: This study was conducted in 3 parts: 1) prevention and 2) reversal of delayed decreases in cerebral perfusion via either intrathecal or intravenous administration of a CPP linked to phosphopeptide mimetics of HSP20 (AZX100) and 3) determining the effect of intravenous administration of AZX100 on blood pressure and heart rate. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Subsequently, AZX100 was administered intrathecally via a cisternal catheter or intravenously. Cerebral perfusion was determined by laser Doppler monitoring. Blood pressure was monitored by telemetry in a separate group of naïve animals treated with AZX100 for 24 hours. RESULTS: The maximal decrease in cerebral perfusion occurred 3 days after SAH. Cisternal administration of AZX100 (0.14-0.57 mg/kg) 24 hours after hemorrhage prevented decreases in cerebral perfusion after SAH. Animals receiving lower doses of AZX100 (0.068 mg/kg) or a scrambled sequence of the active HSP20 peptide linked to CPP developed decreases in cerebral perfusion similar to those seen in control animals. Intravenous administration of AZX100 (1.22 mg/kg) 24 hours after hemorrhage prevented the decreases in cerebral perfusion seen in the controls. Intravenous administration (0.175 mg/kg and 1.22 mg/kg) of AZX100 on Days 2 and 3 after SAH reversed decreases in cerebral perfusion as early as Day 3. There was no impact of AZX100 on blood pressure or heart rate at doses up to 2.73 mg/kg. CONCLUSIONS: Cisternal administration of AZX100 24 hours after hemorrhage prevented decreases in cerebral perfusion. Intravenous administration of AZX100 also prevented and reversed decreases in cerebral perfusion at doses that did not induce hypotension. Transduction of biologically active motifs of downstream regulators like HSP20 represents a potential novel treatment for SAH.

The small heat shock protein, HSPB6, in muscle function and disease.

The small heat shock protein, HSPB6, is a 17-kDa protein that belongs to the small heat shock protein family. HSPB6 was identified in the mid-1990s when it was recognized as a by-product of the purification of HSPB1 and HSPB5. HSPB6 is highly and constitutively expressed in smooth, cardiac, and skeletal muscle and plays a role in muscle function. This review will focus on the physiologic and biochemical properties of HSPB6 in smooth, cardiac, and skeletal muscle; the putative mechanisms of action; and therapeutic implications.

Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-beta1-induced CTGF expression in keloid fibroblasts.

A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

Enhanced skin penetration of P20 phosphopeptide using protein transduction domains.

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent.

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation.

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli.

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

Comparative study of the skin penetration of protein transduction domains and a conjugated peptide.

PURPOSE: We examined the ability of a protein transduction domain (PTD), YARA, to penetrate in the skin and carry a conjugated peptide, P20. The results with YARA were compared to those of a well-known PTD (TAT) and a control, nontransducing peptide (YKAc). The combined action of PTDs and lipid penetration enhancers was also tested. METHODS: YARA, TAT, YKAc, P20, YARA-P20, and TAT-P20 were synthesized by Fmoc chemistry. Porcine ear skin mounted in a Franz diffusion cell was used to assess the topical and transdermal delivery of fluorescently tagged peptides in the presence or absence of lipid penetration enhancers (monoolein or oleic acid). The peptide concentrations in the skin (topical delivery) and receptor phase (transdermal delivery) were assessed by spectrofluorimetry. Fluorescence microscopy was used to visualize the peptides in different skin layers. RESULTS: YARA and TAT, but not YKAc, penetrated abundantly in the skin and permeated modestly across this tissue. Monoolein and oleic acid did not enhance the topical and transdermal delivery of TAT or YARA but increased the topical delivery of YKAc. Importantly, YARA and TAT carried a conjugated peptide, P20, into the skin, but the transdermal delivery was very small. Fluorescence microscopy confirmed that free and conjugated PTDs reached viable layers of the skin. CONCLUSIONS: YARA and TAT penetrate in the porcine ear skin in vitro and carry a conjugated model peptide, P20, with them. Thus, the use of PTDs can be a useful strategy to increase topical delivery of peptides for treatment of cutaneous diseases.

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

Transducible recombinant small heat shock-related protein, HSP20, inhibits vasospasm and platelet aggregation.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstruction. Injury during harvest leads to vasospasm and a thrombogenic endoluminal surface. A proteomic transduction approach was developed to prevent vein graft vasospasm and thrombosis. METHODS: Recombinant HSP20 protein linked to the TAT protein transduction domain was generated in a bacterial expression system (TAT-HSP20). The effect of this protein on the inhibition of smooth muscle contraction was determined using rings of rabbit aorta and HSV in a muscle bath. In addition, the effects of TAT-HSP20 on platelet aggregation were determined in vitro using human citrated whole blood. RESULTS: Recombinant TAT-HSP20 inhibited norepinephrine-induced contraction of rabbit aortic and HSV segments. Similarly, TAT-HSP20 induced smooth muscle relaxation in HSV segments precontracted with norepinephrine. In human-citrated whole blood, platelet aggregation was significantly inhibited by TAT-HSP20 in a dose-dependent manner. CONCLUSIONS: The results of this study demonstrate that recombinant TAT-HSP20 inhibits vascular smooth muscle contraction and platelet aggregation. This suggests that HSP20 may be an ideal effector molecule to target as a proteomic approach to enhance early vein graft patency rates by preventing acute vasospasm and thrombosis.

Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstructions. However, vasospasm can lead to early graft failure. The leading cause of delayed graft failure is intimal hyperplasia. OBJECTIVE: To develop a proteomic approach to prevent vein-graft spasm and intimal hyperplasia. METHODS: Biomimetic peptide analogs of the small heat shock-related protein HSP20, containing a protein transduction domain (PTD), a phosphorylated serine, and a sequence of HSP20 surrounding the phosphorylation site (PTD-pHSP20), or a scrambled sequence of the same amino acids surrounding the phosphorylation site (PTD-scHSP20) were synthesized. The peptides were used in muscle bath and organ culture experiments with human saphenous vein (HSV) segments. Cultured smooth muscle cell lines were used to determine the effect of the peptides on proliferation and migration. RESULTS: In HSV rings precontracted with norepinephrine, PTD-pHSP20 but not PTD-scHSP20 led to relaxation. There was no significant difference in smooth muscle cell proliferation in cells treated with PTD-pHSP20 compared with PTD-scHSP20. Treatment with PTD-pHSP20 significantly inhibited cellular migration compared with PTD-scHSP20. Control, untreated, and PTD-scHSP20-treated saphenous veins had significant increases in intimal thickness after culture. This intimal thickening was completely inhibited by treatment with PTD-pHSP20. CONCLUSIONS: Protein transduction of biologically active motifs of HSP20 can affect pathologic and physiologic responses of HSV and represents a novel proteomic-based therapeutic approach. CLINICAL RELEVANCE: We have been a part of the genomics era and are now viewing the emergence of "proteomics." The genome is linear and relatively easy to examine; however the proteome is much more complex and dynamic. In essence, the purpose of gene therapy is to manipulate the genome to produce a particular protein. This manuscript describes a new proteomic approach in which the biologically active part of a protein is directly introduced into vascular cells. Peptides were synthesized which contained a total of 24 amino acids, 11 of which represent a protein transduction domain or "carrier" while the other 13 are the biologically active "cargo." These synthetic peptides prevent spasm (contraction) and intimal hyperplasia in segments of human saphenous vein treated ex vivo. Preclinical development is currently underway to develop these molecules as a proteomic-based vein harvest solution to enhance vein-graft patency.