Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel.

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Full-length Dhh and N-terminal Shh act as competitive antagonists to regulate angiogenesis and vascular permeability.

AIMS: The therapeutic potential of Hedgehog (Hh) signalling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signalling in vascular biology remain poorly understood. The purpose of the present article is to clarify some conflicting literature data. METHODS AND RESULTS: With this goal, we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hh (N-Shh) and endogenous endothelial-derived Desert Hh (Dhh) induce opposite effects in endothelial cells (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 (Ptch1) demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Ptch1, they induce distinct Ptch1 localization. Finally, we confirmed that in a pathophysiological setting, i.e. brain inflammation, astrocyte-derived N-Shh acts as a FL-Dhh antagonist. CONCLUSION: The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs.

Activation of the Hedgehog Pathway Promotes Recovery of Neurological Function After Traumatic Brain Injury by Protecting the Neurovascular Unit.

The homeostasis of the neurovascular unit (NVU) is disrupted after traumatic brain injury (TBI), and therapeutic strategies targeting the NVU would likely improve neurological outcomes after TBI. Sonic Hedgehog (Shh), which is an endogenous activator of the Hedgehog pathway, promotes brain repair in various injuries. In this study, the controlled cortical impact (CCI) was used to establish a moderate TBI model in adult male Sprague-Dawley rats (250-300 g), and the NVU was reconstructed in vitro from the blood-brain barrier (BBB) and neurons to investigate the effects of exogenous Shh protein on TBI. The modified neurological severity scores (mNSS) and Morris water maze tests were used to evaluate the effect of Shh on neurological function after TBI. The effect of Shh on the NVU in vivo was evaluated by detecting the degrees of cerebral edema and neuronal apoptosis. The integrity and permeability of the BBB, the level of inflammatory factors, and the expression of apoptotic proteins were detected to explore the effect of exogenous Shh on the NVU in vitro. The results showed that exogenous Shh reduced cerebral edema and neuronal apoptosis and promoted neurological recovery after TBI in rats. In vitro experiments showed that Shh-induced activation of the Hedgehog pathway promoted stability of the NVU by reducing damage to the tight junction structure and inhibiting the release of inflammatory factors and neuron apoptosis. Based on these results, the Shh-induced activation of the Hedgehog pathway might be a new promising treatment for TBI.

Optimization of Sonic Hedgehog Delivery to the Penis from Self-Assembling Nanofiber Hydrogels to Preserve Penile Morphology after Cavernous Nerve Injury.

Erectile dysfunction (ED) is a significant medical condition, with high impact on patient quality of life. Current treatments are minimally effective in prostatectomy, diabetic and aging patients due to injury to the cavernous nerve (CN); loss of innervation causes extensive smooth muscle (SM) apoptosis, increased collagen and ED. Sonic hedgehog (SHH) is a critical regulator of penile SM. We developed a self-assembling peptide amphiphile (PA) nanofiber hydrogel for extended release of SHH protein to the penis after CN injury, to suppress SM apoptosis. In this study we optimize the animal model, SHH concentration, duration of suppression, and location of delivery, to maximize SM preservation. SHH treatment suppressed apoptosis and preserved SM 48%. Increased SHH duration preserved SM 100%. Simultaneous penis/CN delivery increased SM 127%. Optimization of SHH PA delivery is essential for clinical translation to ED patients, and the PA vehicle has wide applicability as an in vivo delivery tool.

Peptide amphiphile delivery of sonic hedgehog protein promotes neurite formation in penile projecting neurons.

Erectile dysfunction (ED) critically impacts quality of life in prostatectomy, diabetic and aging patients. The underlying mechanism involves cavernous nerve (CN) damage, resulting in ED in 80% of prostatectomy patients. Peptide amphiphile (PA) nanofiber hydrogel delivery of sonic hedgehog (SHH) protein to the injured CN, improves erectile function by 60% at 6 weeks after injury, by an unknown mechanism. We hypothesize that SHH is a regulator of neurite formation. SHH treatment promoted extensive neurite formation in uninjured and crushed CNs, and SHH inhibition decreased neurites >80%. Most abundant neurites were observed with continuous SHH PA treatment of crushed CNs. Once induced with SHH, neurites continued to grow. SHH rescued neurite formation when not given immediately. SHH is a critical regulator of neurite formation in peripheral neurons under uninjured and regenerative conditions, and SHH PA treatment at the time of injury/prostatectomy provides an exploitable avenue for intervention to prevent ED.

Genetic detection of Sonic hedgehog (Shh) expression and cellular response in the progression of acute through chronic demyelination and remyelination.

Multiple sclerosis is a demyelinating disease in which neurological deficits result from damage to myelin, axons, and neuron cell bodies. Prolonged or repeated episodes of demyelination impair remyelination. We hypothesized that augmenting Sonic hedgehog (Shh) signaling in chronically demyelinated lesions could enhance oligodendrogenesis and remyelination. Shh regulates oligodendrocyte development during postnatal myelination, and maintains adult neural stem cells. We used genetic approaches to detect Shh expression and Shh responding cells in vivo. ShhCreERT2 or Gli1CreERT2 mice were crossed to reporter mice for genetic fate-labeling of cells actively transcribing Shh or Gli1, an effective readout of canonical Shh signaling. Tamoxifen induction enabled temporal control of recombination at distinct stages of acute and chronic cuprizone demyelination of the corpus callosum. Gli1 fate-labeled cells were rarely found in the corpus callosum with tamoxifen given during acute demyelination stages to examine activated microglia, reactive astrocytes, or remyelinating cells. Gli1 fate-labeled cells, mainly reactive astrocytes, were observed in the corpus callosum with tamoxifen given after chronic demyelination. However, Shh expressing cells were not detected in the corpus callosum during acute or chronic demyelination. Finally, SAG, an agonist of both canonical and type II non-canonical Hedgehog signaling pathways, was microinjected into the corpus callosum after chronic demyelination. Significantly, SAG delivery increased proliferation and enhanced remyelination. SAG did not increase Gli1 fate-labeled cells in the corpus callosum, which may indicate signaling through the non-canonical Hedgehog pathway. These studies demonstrate that Hedgehog pathway interventions may have therapeutic potential to modulate astrogliosis and to promote remyelination after chronic demyelination.

Delivery of Sonic Hedgehog Gene Repressed Irradiation-induced Cellular Senescence in Salivary Glands by Promoting DNA Repair and Reducing Oxidative Stress.

Rationale: Irreversible hypofunction of salivary glands or xerostomia is common in head and neck cancer survivors treated with radiotherapy even when various new techniques are applied to minimize the irradiation (IR) damage. This condition severely impairs the quality of life of patients and can only be temporarily relieved with current treatments. We found recently that transient expression of Sonic Hedgehog (Shh) in salivary glands after IR rescued salivary function, but the underlying mechanisms are not totally clear. Methods: We generated a mouse model of IR-induced hyposalivation, and delivered adenoviral vectors carrying Shh or control GFP gene into submandibular glands (SMGs) via retrograde ductal instillation 3 days after IR. The cellular senescence was evaluated by senescence-associated beta-galactosidase assay and the expression of senescence markers. The underlying mechanisms were explored by examining DNA damage, oxidative stress, and the expression of related genes by qRT-PCR, Western blot and immunofluorescent staining. Results: Shh gene transfer repressed IR-induced cellular senescence by promoting DNA repair and decreasing oxidative stress, which is mediated through upregulating expression of genes related to DNA repair such as survivin and miR-21 and repressing expression of pro-senescence gene Gdf15 likely downstream of miR-21. Conclusion: Repressing cellular senescence contributes to the rescue of IR-induced hyposalivation by transient activation of Hh signaling, which is related to enhanced DNA repair and decreased oxidative stress in SMGs.

Poststroke Sonic Hedgehog Agonist Treatment Improves Functional Recovery by Enhancing Neurogenesis and Angiogenesis.

BACKGROUND AND PURPOSE: Because of the limitation in treatment window of the r-tPA (recombinant tissue-type plasminogen activator), the development of delayed treatment for stroke is needed. In this study, we examined the efficacy of delayed poststroke treatment (post 3-8 days) of the sonic hedgehog pathway agonist on functional recovery and the underlying mechanisms. METHODS: We evaluated functional recovery at 1 month after stroke using locomotion analysis and Barnes maze test for cognitive function. We used a genetically inducible neural stem cell-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track their proliferation, survival, and differentiation in ischemic brain. Brain tissue damage, angiogenesis, and cerebral blood flow recovery was evaluated using magnetic resonance imaging techniques and immunostaining. RESULTS: Our results show that delayed treatment of sonic hedgehog pathway agonist in stroke mice results in enhanced functional recovery both in locomotor function and in cognitive function at 1 month after stroke. Furthermore, using the Nestincre-ERT2-YFP mice, we showed that poststroke sonic hedgehog pathway agonist treatment increased surviving newly born cells derived from both subventricular zone and subgranular zone neural stem cells, total surviving DCX+ (Doublecortin) neuroblast cells, and neurons (NeuN+/YFP+) in the ischemic brain. Sonic hedgehog pathway agonist treatment also improved the brain tissue repair in ischemic region supported by our T2-weighted magnetic resonance imaging, cerebral blood flow map by arterial spin labeling, and immunohistochemistry (α-smooth muscle actin and CD31 immunostaining). CONCLUSIONS: These data confirm an important role for the hedgehog pathway in poststroke brain repair and functional recovery, suggesting a prolonged treatment window for potential treatment strategy to modulate sonic hedgehog pathway after stroke.

Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing.

BACKGROUND: Nonhealing bone defects represent an immense biomedical burden. Despite recent advances in protein-based bone regeneration, safety concerns over bone morphogenetic protein-2 have prompted the search for alternative factors. Previously, the authors examined the additive/synergistic effects of hedgehog and Nel-like protein-1 (NELL-1) on the osteogenic differentiation of mesenchymal stem cells in vitro. In this study, the authors sought to leverage their previous findings by applying the combination of Smoothened agonist (SAG), hedgehog signal activator, and NELL-1 to an in vivo critical-size bone defect model. METHODS: A 4-mm parietal bone defect was created in mixed-gender CD-1 mice. Treatment groups included control (n = 6), SAG (n = 7), NELL-1 (n = 7), and SAG plus NELL-1 (n = 7). A custom fabricated poly(lactic-co-glycolic acid) disk with hydroxyapatite coating was used as an osteoinductive scaffold. RESULTS: Results at 4 and 8 weeks showed increased bone formation by micro-computed tomographic analyses with either stimulus alone (SAG or NELL-1), but significantly greater bone formation with both components combined (SAG plus NELL-1). This included greater bone healing scores and increased bone volume and bone thickness. Histologic analyses confirmed a significant increase in new bone formation with the combination therapy SAG plus NELL-1, accompanied by increased defect vascularization. CONCLUSIONS: In summary, the authors' results suggest that combining the hedgehog signaling agonist SAG and NELL-1 has potential as a novel therapeutic strategy for the healing of critical-size bone defects. Future directions will include optimization of dosage and delivery strategy for an SAG and NELL-1 combination product.

Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy.

Hedgehog pathway mediates early acceleration of liver regeneration induced by a novel two-staged hepatectomy in mice.

BACKGROUND & AIMS: ALPPS, a novel two-staged approach for the surgical removal of large/multiple liver tumors, combines portal vein ligation (PVL) with parenchymal transection. This causes acceleration of compensatory liver growth, enabling faster and more extensive tumor removal. We sought to identify the plasma factors thought to mediate the regenerative acceleration following ALPPS. METHODS: We compared a mouse model of ALPPS against PVL and additional control surgeries (n=6 per group). RNA deep sequencing was performed to identify candidate molecules unique to ALPPS liver (n=3 per group). Recombinant protein and a neutralizing antibody combined with appropriate surgeries were used to explore candidate functions in ALPPS (n=6 per group). Indian hedgehog (IHH/Ihh) levels were assessed in human ALPPS patient plasma (n=6). RESULTS: ALPPS in mouse confirmed significant acceleration of liver regeneration relative to PVL (p<0.001). Ihh mRNA, coding for a secreted ligand inducing hedgehog signaling, was uniquely upregulated in ALPPS liver (p<0.001). Ihh plasma levels rose 4h after surgery (p<0.01), along with hedgehog pathway activation and subsequent cyclin D1 induction in the liver. When combined with PVL, Ihh alone was sufficient to induce ALPPS-like acceleration of liver growth. Conversely, blocking Ihh markedly inhibited the accelerating effects of ALPPS. In the small cohort of ALPPS patients, IHH tended to be elevated early after surgery. CONCLUSIONS: Ihh and hedgehog pathway activation provide the first mechanistic insight into the acceleration of liver regeneration triggered by ALPPS surgery. The accelerating potency of recombinant Ihh, and its potential effect in human ALPPS may lead to a clinical role for this protein. LAY SUMMARY: ALPPS, a novel two-staged hepatectomy, accelerates liver regeneration, thereby helping to treat patients with otherwise unresectable liver tumors. The molecular mechanisms behind this accelerated regeneration are unknown. Here, we elucidate that Indian hedgehog, a secreted ligand important for fetal development, is a crucial mediator of the regenerative acceleration triggered by ALPPS surgery.

Sonic hedgehog delivery from self-assembled nanofiber hydrogels reduces the fibrotic response in models of erectile dysfunction.

Erectile dysfunction (ED) is a serious medical condition in which current treatments are ineffective in prostatectomy and diabetic patients, due to injury to the cavernous nerve (CN), which causes irreversible remodeling of the penis (decreased smooth muscle and increased collagen), through a largely undefined mechanism. We propose that sonic hedgehog (SHH) and neural innervation, are indispensable regulators of collagen in the penis, with decreased SHH protein being an integral component of the fibrotic response to loss of innervation. We examined collagen abundance and morphology in control (Peyronie's), prostatectomy and diabetic patients, and in rat models of penile development, CN injury, SHH inhibition and under regenerative conditions, utilizing self-assembling peptide amphiphile (PA) nanofiber hydrogels for SHH delivery. Collagen abundance increased in penis of ED patients. In rats, collagen increased with CN injury in a defined time frame independent of injury severity. An inverse relationship between SHH and collagen abundance was identified; SHH inhibition increased and SHH treatment decreased penile collagen. SHH signaling in the pelvic ganglia (PG)/CN is important to maintain CN integrity and when inhibited, downstream collagen induction occurs. Collagen increased throughout penile development and with age, which is important when considering how to treat fibrosis clinically. These studies show that SHH PA treatment reduces collagen under regenerative post-prostatectomy conditions, indicating broad application for ED prevention in prostatectomy, diabetic and aging patients and in other peripheral nerve injuries. The PA nanofiber protein vehicle may be widely applicable as an in vivo delivery tool. STATEMENT OF SIGNIFICANCE: We use self-assembling peptide amphiphiles (PA) as biological delivery vehicles to prevent cavernous nerve (CN) injury induced erectile dysfunction (ED). These versatile hydrogels were molecularly pre-programmed for sonic hedgehog (SHH) protein delivery, either from an injectable solution with fast, in situ assembly into a soft hydrogel, or by highly aligned monodomain nanofiber bundles. We used PAs to examine a novel neuronal component to collagen regulation and the role of SHH in the fibrotic response to CN injury. SHH perturbation in the penis or the CN, selectively impacts collagen, with SHH inhibition increasing and SHH treatment suppressing collagen. These results suggest that SHH treatment by PA has translational potential to suppress collagen induction and remodelling, an irreversible component of ED development.

Controlled Delivery of Sonic Hedgehog with a Heparin-Based Coacervate.

Here we describe the preparation of a delivery vehicle for controlled release of Sonic hedgehog (Shh). The vehicle contains a synthetic polycation and heparin which interact by polyvalent charge attraction and rapidly self-assemble into liquid coacervate droplets. The coacervate loads Shh with high efficiency, protects its bioactivity, and provides sustained and localized release at the site of application. Shh coacervate may be injected directly or coated onto a polymeric scaffold for tissue engineering approaches, as described here.

Activation of Sonic Hedgehog Leads to Survival Enhancement of Astrocytes via the GRP78-Dependent Pathway in Mice Infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytes may protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh) via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infected BABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated with corresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP) and Shh were increased in astrocyte cocultured with living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated in cocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higher in astrocytes pretreated with recombinant Shh. These findings suggest that the expression of Shh may inhibit cell death by activating Bcl-2 through a GRP78-dependent pathway.

Exogenous Sonic hedgehog modulates the pool of GABAergic interneurons during cerebellar development.

All cerebellar GABAergic interneurons were derived from a common pool of precursor cells residing in the embryonic ventricular zone (VZ) and migrating in the prospective white matter (PWM) after birth, where both intrinsic and extrinsic factors contribute to regulate their amplification. Among the environmental factors, we focused on Sonic hedgehog (Shh), a morphogen well known to regulate neural progenitor cell proliferation. We asked if and how exogenous Shh treatment affects the lineage of cerebellar GABAergic interneurons. To address these issues, exogenous Shh was administered to embryonic and postnatal organotypic slices. We found that Shh is able to expand the pool of interneuron progenitors residing in the embryonic epithelium and in the postnatal PWM. In particular, Shh signalling pathway was highly mitogenic at early developmental stages of interneuron production, whereas its effect decreased after the first postnatal week. Gene expression analysis of sorted cells and in situ hybridization further showed that immature interneurons express both the Shh receptor patched and the Shh target gene Gli1. Thus, within the interneuron lineage, Shh might exert regulatory functions also in postmitotic cells. On the whole, our data enlighten the role of Shh during cerebellar maturation and further broaden our knowledge on the amplification mechanisms of the interneuron progenitor pool.

Effects of exogenous sonic hedgehog peptide on proliferation, adhesion, migration of endothelial progenitor cells from peripheral blood.

In order to study the effects of exogenous sonic hedgehog (shh) peptide on proliferation, adhesion, migration of endothelial progenitor cells (EPCs) from rat peripheral blood, the mononuclear cells were collected from rat peripheral blood by Ficoll density gradient centrifugation. EPCs were isolated with adherence screening method and cultured in M199 culture medium with the supplement of VEGF and bFGF. The immunohistochemical staining was used to identify cell markers such as CDl33 and VEGFR-2. EPCs were stimulated with exogenous shh peptide of different final concentrations (0.01, 0.1, 1, 10 µg/mL). The proliferation, adhesion and migration of EPCs were detected by MTT chromometry, adhesion test and transwell system, respectively. The results of this study showed that, after 7 days of culture, cells formed clusters, assuming typical cobbles-tone pattern under microscope. After 2 weeks of culture, cells were arranged in cord-like fashion and sometimes grew like "micro-vessels". Immunohistochemical staining showed that the cultured cells were positive for both CD133 and VEGFR-2. The proliferation, adhesion and migration of EPCs could be promoted by endogenous shh peptide at concentrations from 0.1 µg/mL to 10 µg/mL in a concentration-dependent manner. The findings indicate that exogenous shh peptide can enhance EPCs proliferation, adhesion, and migration, which may have a potential value for clinical application.

Controlled delivery of sonic hedgehog morphogen and its potential for cardiac repair.

The morphogen Sonic hedgehog (Shh) holds great promise for repair or regeneration of tissues suffering ischemic injury, however clinical translation is limited by its short half-life in the body. Here, we describe a coacervate delivery system which incorporates Shh, protects it from degradation, and sustains its release for at least 3 weeks. Shh released from the coacervate stimulates cardiac fibroblasts to upregulate the expression of multiple trophic factors including VEGF, SDF-1α, IGF-1, and Shh itself, for at least 48 hours. Shh coacervate also demonstrates cytoprotective effects for cardiomyocytes in a hydrogen peroxide-induced oxidative stress environment. In each of these studies the bioactivity of the Shh coacervate is enhanced compared to free Shh. These results warrant further investigation of the in vivo efficacy of Shh coacervate for cardiac repair.

Sonic hedgehog-modified human CD34+ cells preserve cardiac function after acute myocardial infarction.

RATIONALE: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. OBJECTIVE: To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). METHODS AND RESULTS: When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. CONCLUSIONS: Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.

Improvement of neurological recovery and stimulation of neural progenitor cell proliferation by intrathecal administration of Sonic hedgehog.

OBJECT: Sonic hedgehog (Shh) is a glycoprotein molecule that has been shown to be associated with the proliferative capacity of endogenous neural precursor cells during embryonic development. It has also been shown to regulate the proliferative capacity of neural stem cells in the adult subventricular zone (SVZ), which are also upregulated in animal models of ischemic stroke. In the present study, the effects of exogenous administration of intrathecal Shh protein were examined in the setting of a rodent model of ischemic stroke, with particular attention given to endogenous neural stem cell proliferation and migration as well as inducible differences in behavioral recovery. METHODS: A rodent model of ischemic stroke was created using the intraluminal suture method of reversible middle cerebral artery occlusion. Animals were treated with intrathecal administration of Shh protein at 24 hours after the onset of the stroke. Behavioral testing was performed, and the animals were killed for measurements of infarct volume 7 days after stroke. Immunohistochemical staining was performed and measurements of cellular proliferation were obtained, with a focus on the proportion and distribution of neural progenitor cells in the SVZ. These values were compared across experimental groups. RESULTS: Treatment with intrathecal Shh protein resulted in significant improvement in behavioral function compared with the control group, with a significant reduction of ischemic tissue in the cerebral hemisphere. An increase of nestin immunoreactive cells was observed along the SVZ. CONCLUSIONS: Intrathecal Shh agonist at doses that upregulate spinal cord GLI1 transcription increases the population of neural precursor cells after spinal cord injury in adult rats. Intrathecal administration of Shh protein appears to have a neuroprotective effect in animal models of ischemic stroke and is associated with improved behavioral recovery, which may be related to its effects on neurogenesis in the SVZ and could be associated with improved functional recovery.