Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery After Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the periventricular white matter, which results in arrested maturation of OPCs and myelination failure. HA is a major component of the extracellular matrix of the brain, which regulates inflammation through CD44 and TLR2/4 receptors. Here, we show two mechanism-based strategies that effectively enhanced myelination and neurological recovery in preterm rabbit model of IVH. First, degrading HA by hyaluronidase treatment reduced CD44 and TLR4 expression, proinflammatory cytokines, and microglial infiltration, as well as promoted oligodendrocyte maturation and myelination. Second, intraventricular injection of HA oligosaccharide reduced inflammation and enhanced myelination, conceivably by depleting HC-HA levels

Bone Morphogenetic Protein Inhibition Promotes Neurological Recovery after Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) results in neural cell death and white matter injury in premature infants. No therapeutic strategy is currently available against this disorder. Bone morphogenetic protein (BMP) signaling suppresses oligodendrocyte development through basic-helix-loop-helix (bHLH) transcription factors and promotes astrocytosis. Therefore, we hypothesized that IVH in premature newborns initiates degeneration and maturation arrest of oligodendrocyte lineage and that BMP inhibition alleviates hypomyelination, gliosis, and motor impairment in the survivors of IVH. To test the hypotheses, a rabbit model of IVH was used in which premature rabbit pups (E29) are treated with intraperitoneal glycerol at 2 hours of age to induce IVH; and the pups with IVH exhibit hypomyelination and gliosis at 2 weeks of postnatal age. Maturation of oligodendrocyte lineage was evaluated by specific markers, and the expression of bHLH transcription factors was assessed. BMP levels were measured in both premature rabbit pups and autopsy materials from premature infants. Recombinant human noggin was used to suppress BMP action; and neurobehavioral performance, myelination and gliosis were assessed in noggin-treated pups compared with untreated controls. We found that IVH resulted in apoptosis and reduced proliferation of oligodendrocyte progenitors, as well as arrested maturation of preoligodendrocytes in rabbits. BMP4 levels were significantly elevated in both rabbit pups and human premature infants with IVH compared with controls. Importantly, BMP inhibition by recombinant human noggin restored the levels of phospho-Smad1/5/8, Olig2 transcription factor, oligodendrocyte maturation, myelination, astrocyte morphology, and motor function in premature pups with IVH. Hence, BMP inhibition might enhance neurological recovery in premature infants with IVH

Emerging Therapies for Brain Recovery After IVH in Neonates: Cord Blood Derived Mesenchymal Stem Cells (MSC) and Unrestricted Somatic Stem Cells (USSC)

In this report, we summarize evidence on mechanisms of injury after intraventricular hemorrhage resulting in post-hemorrhagic white matter injury and hydrocephalus and correlate that with the possibility of cellular therapy. We describe how two stem cell lines (MSC & USSC) acting in a paracrine fashion offer promise for attenuating the magnitude of injury in animal models and for improved functional recovery by: lowering the magnitude of apoptosis and neuronal cell death, reducing inflammation, and thus, mitigating white matter injury that culminates in improved motor and neurocognitive outcomes. Animal models of IVH are analyzed for their similarity to the human condition and we discuss merits of each approach. Studies on stem cell therapy for IVH in human neonates is described. Lastly, we offer suggestions on what future studies are needed to better understand mechanisms of injury and recovery and argue that human trials need to be expanded in parallel to animal research

A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

The human angiotensinogen (hAGT) gene contains an A/G polymorphism at −217, and frequency of −217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant −217A almost always occurs with −532T, −793A, and −1074T, whereas variant −217G almost always occurs with −532C, −793G, and −1074G. Since allele −6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, −6A:−217A (AA) and −6A:−217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by ∼40%, and plasma angiotensin II level is increased by ∼50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure

Scn1b expression in the adult mouse heart modulates Na⁺ influx in myocytes and reveals a mechanistic link between Na⁺ entry and diastolic function

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na(+) current (I(Na)) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of I(Na). Variants in the gene SCN1B, encoding the VGSC β1- and β1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of β1/β1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of I(Na), with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late I(Na) with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca(2+) transients and cell mechanics, defects that were corrected by inhibition of I(Na). Collectively, these results document that VGSC β1/β1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na(+) influx in cardiomyocytes. NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b, the gene encoding voltage-gated sodium channel β1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na(+) influx and Ca(2+) cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of β1-subunits in the physiology of the heart

Early Postnatal Expression of Tgfβ-1 and Fgf-2 Correlates With Regenerative Functions of Unrestricted Somatic Stem Cell Infusion After Rabbit GMH-IVH

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth associated with white matter injury (WMI) and reduced neurogenesis. IVH commonly arises from the germinal matrix, a highly cellular, transient structure, where all precursor cells are born, proliferate, and migrate during brain development. IVH leads to reduced progenitor cell proliferation and maturation and contributes to WMI. Interruption of oligodendrocyte lineage (OL) proliferation and maturation after IVH will prevent myelination. We evaluated whether unrestricted somatic stem cells (USSCs) could recover OL lineage, as USSC release multiple relevant growth factors and cytokines. The effects of USSC infusion at 24 hours after IVH were assessed in the periventricular zone by analysis of OL lineage-specific progression (PDGFR+, OLIG2+, NKX2.2+ with Ki67), and this was correlated with growth factors TGFβ1, FGF2 expression. The early OL cell lineage by immunofluorescence and cell density quantitation showed significant reduction after IVH (P \u3c .05 both PDGFR+, OLIG2+ at day 3); with significant recovery after injection of USSCs (P \u3c .05 both PDGFR+, OLIG2+ at day 3). CSF protein and tissue mRNA levels of TGFβ1 were reduced by IVH and recovered after USSC (P \u3c .05 for all changes). FGF2 showed an increased mRNA after USSC on day3 (P \u3c .05). Cell cyclin genes were unaffected except for the cycle inhibitor P27Kip1 which increased after IVH but returned to normal after USSC on day 3. Our findings demonstrated a plausible mechanism through which USSCs can aid in developmental myelination by recovery of OL proliferation and maturation along with correlative changes in growth factors during brain development

Human Cord Blood-Derived Unrestricted Somatic Stem Cell Infusion Improves Neurobehavioral Outcome in a Rabbit Model of Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth, which leads to hydrocephalus, cerebral palsy, and mental retardation. There are no available therapies to cure IVH and standard treatment is supportive care. Unrestricted somatic stem cells (USSCs) from human cord blood have reparative effects in animal models of brain and spinal cord injuries. USSCs were administered to premature rabbit pups with IVH and their effects on white matter integrity and neurobehavioral performance were evaluated. USSCs were injected either via intracerebroventricular (ICV) or via intravenous (IV) routes in 3 days premature (term 32d) rabbit pups, 24 hours after glycerol-induced IVH. The pups were sacrificed at postnatal days 3, 7, and 14 and effects were compared to glycerol-treated but unaffected or nontreated control. Using in vivo live bioluminescence imaging and immunohistochemical analysis, injected cells were found in the injured parenchyma on day 3 when using the IV route compared to ICV where cells were found adjacent to the ventricle wall forming aggregates; we did not observe any adverse events from either route of administration. The injected USSCs were functionally associated with attenuated microglial infiltration, less apoptotic cell death, fewer reactive astrocytes, and diminished levels of key inflammatory cytokines (TNFalpha and IL1beta). In addition, we observed better preservation of myelin fibers, increased myelin gene expression, and altered reactive astrocyte distribution in treated animals, and this was associated with improved locomotor function. Overall, our findings support the possibility that USSCs exert anti-inflammatory effects in the injured brain mitigating many detrimental consequences associated with IVH. Stem Cells Translational Medicine 2019

Human Cord Blood Derived Unrestricted Somatic Stem Cells Restore Aquaporin Channel Expression, Reduce Inflammation and Inhibit the Development of Hydrocephalus After Experimentally Induced Perinatal Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) is a severe complication of preterm birth associated with cerebral palsy, intellectual disability, and commonly, accumulation of cerebrospinal fluid (CSF). Histologically, IVH leads to subependymal gliosis, fibrosis, and disruption of the ependymal wall. Importantly, expression of aquaporin channels 1 and 4 (AQP1 and AQP4) regulating respectively, secretion and absorption of cerebrospinal fluids is altered with IVH and are associated with development of post hemorrhagic hydrocephalus. Human cord blood derived unrestricted somatic stem cells (USSCs), which we previously demonstrated to reduce the magnitude of hydrocephalus, as having anti-inflammatory, and beneficial behavioral effects, were injected into the cerebral ventricles of rabbit pups 18 h after glycerol-induced IVH. USSC treated IVH pups showed a reduction in ventricular size when compared to control pups at 7 and 14 days (both, P \u3c 0.05). Histologically, USSC treatment reduced cellular infiltration and ependymal wall disruption. In the region of the choroid plexus, immuno-reactivity for AQP1 and ependymal wall AQP4 expression were suppressed after IVH but were restored following USSC administration. Effects were confirmed by analysis of mRNA from dissected choroid plexus and ependymal tissue. Transforming growth factor beta (TGF-β) isoforms, connective tissue growth factor (CTGF) and matrix metalloprotease-9 (MMP-9) mRNA, as well as protein levels, were significantly increased following IVH and restored towards normal with USSC treatment (P \u3c 0.05). The anti-inflammatory cytokine Interleukin-10 (IL-10) mRNA was reduced in IVH, but significantly recovered after USSC injection (P \u3c 0.05). In conclusion, USSCs exerted anti-inflammatory effects by suppressing both TGF-β specific isoforms, CTGF and MMP-9, recovered IL-10, restored aquaporins expression towards baseline, and reduced hydrocephalus. These results support the possibility of the use of USSCs to reduce IVH consequences in prematurity

The mutational spectrum of brachydactyly type C

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5