Abstract It is challenging to regenerate periodontal tissues fully. We have previously reported a heparan sulfate variant with enhanced affinity for bone morphogenetic protein-2, termed HS3, that enhanced periodontal tissue regeneration in a rodent model. Here we seek to transition this work closer to the clinic and investigate the efficacy of the combination HS3 collagen device in a non-human primate (NHP) periodontitis model. Wire-induced periodontitis was generated in ten Macaca fascicularis, and defects were treated with Emdogain or collagen (CollaPlug) loaded with (1) distilled water, (2) HS low (36 µg of HS3), or (3) HS high (180 µg of HS3) for 3 months. At the endpoint, microscopic assessment showed significantly less epithelial down-growth, greater alveolar bone filling, and enhanced cementum and periodontal ligament regeneration following treatment with the HS-collagen combination devices. When evaluated using a periodontal regeneration assessment score (PRAS) on a scale of 0–16, collagen scored 6.78 (± 2.64), Emdogain scored 10.50 (± 1.73) and HS low scored 10.40 (± 1.82). Notably, treatment with HS high scored 12.27 (± 2.20), while healthy control scored 14.80 (± 1.15). This study highlights the efficacy of an HS-collagen device for periodontal regeneration in a clinically relevant NHP periodontitis model and warrants its application in clinical trials.
A canal was surgically created in the lower lip of male Sprague-Dawley rats and used as a reservoir for moist snuff. A total of 230 animals were randomized into six groups, five containing 40 rats and one containing 30 rats. After 2 wk of recuperation, the animals were treated as follows. Group I was initiated with 7,12-dimethylbenz(a)anthracene 3 times/wk for 4 wk followed by cotton pellet administration. Group II was initiated with 7,12-dimethylbenz(a)anthracene for 4 wk followed by snuff twice a day, 5 days/wk. Group III received snuff twice a day, 5 days/wk. Groups IV and V were initiated with 4-nitroquinoline N-oxide 3 days/wk for 4 wk. Thereafter Group IV received a cotton pellet, and Group V rats were treated with snuff twice a day, 5 days/wk. Group VI received a cotton pellet once a day, 5 days/wk. Treatment of all groups continued for a maximum of 104 wk. Group V rats had a significantly lower mean survival time than did the other groups because of the development of lip sarcomas in 66% of the rats as compared with 23% in Group II and 26% in Group III. One rat in each of Groups IV and VI developed lip sarcomas. The incidence of sarcomas in Group V as compared with the other groups is statistically significant (P less than 0.05 to 0.001). Spindle cell proliferation, a possible precursor lesion of lip sarcoma, was found in five rats of Group II, seven of Group III, and four of Group V. These results show that snuff has strong promoting capability with regard to the development of lip sarcomas after 4-nitroquinoline N-oxide initiation, but not after 7,12-dimethylbenz(a)anthracene initiation. Snuff by itself caused three squamous carcinomas of the palate, two squamous cell papillomas of the lip, and ten lip sarcomas (in 38 rats as compared with one lip sarcoma in 30 control rats), showing snuff to be carcinogenic for the lip and oral cavity.
Bone morphogenetic protein 2 (BMP-2) is an osteoinductive protein and a potent inducers of bone formation, playing an essential role during bone fracture repair. Heparan sulfate (HS), a highly charged and linear polysaccharide, is known to interact with and enhance BMP-2 bioactivity. Despite showing potential as a potent adjuvant of the endogenous bone healing response, commercially available HS is derived from animal sources which are less desirable when considering translation into the clinic. In the present study, we screen twenty glycomimetics against BMP-2 to determine if fully synthetic analogues of HS can enhance the bioactivity of BMP-2 in vitro and bone healing in vivo. We found that a four-armed dendrimer harboring oversulfated maltose residues could bind BMP-2 with high affinity, enhance BMP-2 bioactivity in vitro and enhance bone regeneration in vivo. These data suggest fully synthetic glycomimetics are viable alternatives to naturally derived HS and offer an attractive alternative for clinical translation.
Growth factor (GF) signaling is a key determinant of stem cell fate. Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs). Here, we report a cell surface engineering strategy that exploits the function of HSPGs to promote differentiation in embryonic stem cells (ESCs). We have generated synthetic neoproteoglycans (neoPGs) with affinity for the fibroblast growth factor 2 (FGF2) and introduced them into plasma membranes of ESCs deficient in HS biosynthesis. There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification. This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.
Sodium saccharin and sodium ascorbate are known to promote urinary bladder carcinogenesis in rats following initiation with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-butyl-N-(4-hydroxybutyl) nitrosamine. Sodium salts of other organic acids have also been shown to be bladder tumor promoters. In addition, these substances increase urothelial proliferation in short term assays in rats when fed at high doses. When they have been tested, the acid forms of these salts are without either promoting or cell proliferative inducing activity. The following experiment was designed to compare the tumor promoting activity of various forms of saccharin and to evaluate the role in promotion of urinary sodium, calcium, and pH as well as other factors. Twenty groups of 40 male F344 rats, 5 weeks of age, were fed either FANFT or control diet during a 6-week initiation phase followed by feeding of a test compound for 72 weeks in the second phase. The chemicals were administered to the first 18 groups in Agway Prolab 3200 diet and the last 2 groups were fed NIH-07 diet. The treatments were as follows: (a) FANFT----5% sodium saccharin (NaS); (b) FANFT----3% NaS; (c) FANFT----5.2% calcium saccharin (CaS); (d) FANFT----3.12% CaS; (e) FANFT----4.21% acid saccharin (S); (f) FANFT----2.53% S; (g) FANFT----5% sodium ascorbate; (h) FANFT----4.44% ascorbic acid; (i) FANFT----5% NaS plus 1.15% CaCO3; (j) FANFT----5.2% CaS plus 1.34% NaCl; (k) FANFT----5% NaS plus 1.23% NH4Cl; (l) FANFT----1.15% CaCO3; (m) FANFT----1.34% NaCl; (n) FANFT----control; (o) control----5% NaS; (p) control----5.2% CaS; (q) control----4.21% S; (r) Control----control; (s) FANFT----5% NaS (NIH-07 diet); (t) FANFT----control (NIH-07 diet). NaS, CaS and S without prior FANFT administration were without tumorigenic activity. NaS was found to have tumor promoting activity, showing a positive response at the 5 and 3% dose levels, with significantly greater activity at the higher dose. CaS had slight tumor promoting activity but without a dose response, and S showed no tumor promoting activity. In addition, NaCl showed weak tumor promoting activity, but CaCO3 was without activity. NH4Cl completely inhibited the tumor promoting activity of NaS when concurrently administered with it. NaCl administered with CaS or CaCO3 administered with NaS showed activity similar to that of NaS. Sodium ascorbate was also shown to have tumor promoting activity, with slightly less activity than NaS. Ascorbic acid showed no tumor promoting activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is upregulated in malignant cancers and inflammation, aiding cell migration and the release of signaling molecules. It is established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations, because of cost, production complexity, or off-target effects. Here, we report the synthesis of a novel, targeted library of single-entity glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM IC50 potency for heparanase inhibition equivalent to comparator compounds in clinical development, and potently inhibit metastasis and growth of human myeloma tumor cells in a mouse xenograft model. Importantly, they lack anticoagulant activity and cytotoxicity, and also inhibit angiogenesis. They provide a new candidate class for anticancer and wider therapeutic applications, which could benefit from targeted heparanase inhibition.
Although VEGF165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF165-binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function.Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed.HS7 significantly enhanced VEGF165-mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity.A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF165 activity during the post-ischemic recovery phase of stroke.
The anesthetic potencies (ED50) of four gaseous anesthetics and five liquid anesthetics were first determined in newts, using the abolition of righting reflex measured by the rolling response at 20°. The following results were obtained: N2O, 0.69 atm; N2, 21.5 atm; SF6, 1.82 atm; CF4, 11.0 atm; CHCl3 0.89 mM; butanol, 16.7 mM; pentobarbitone sodium, 0.85 mM; halothane, 0.39 mM; ether, 25 mM. The ability of elevated pressures to antagonize the effect of these anesthetics was then studied. For the liquid anesthetics, a graded response to pressure was observed and the reversibility of the antagonistic effect was demonstrated. Dose-response curves were obtained for the interaction of pressure with the gaseous anesthetics, and, from these, ED50 values at various pressures have been interpolated. The data are used to compare the Meyer-Overton and the critical volume hypotheses; the latter not only is consistent with the data but also provides explanations for the antagonistic phenomenon and the lack of anesthetic effect for helium, neon, and hydrogen. The critical volume hypothesis is developed for three solvent model systems, from which estimates of the compressibility of the site of action are made.
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