The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.
Clinical and experimental immunology
2016; 185 (3): 372-381
Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis
JOURNAL OF CLINICAL INVESTIGATION
2015; 125 (10): 3928-3940
Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice.
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society
Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan synthesis in mouse models of cancer, autoimmunity, and a variety of other inflammatory disorders where hyaluronan (HA) has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration, and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after one week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a one-week loading period on the drug is required for most protocols. At steady state, over 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulfated metabolite, 4-methylumbelliferyl sulfate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0.65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/cei.12815
View details for PubMedID 27218304
Impact of the Hypoxia-Inducible Factor-1 alpha (HIF1A) Pro582Ser Polymorphism on Diabetes Nephropathy
2013; 36 (2): 415-421
Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired due to the decreased levels of local stromal-derived factor-1? (SDF-1?). Therefore, we investigated the influence of HBO on HIF-1, which is a central regulator of SDF-1? and is down-regulated in diabetic wounds. The effects of HBO on HIF-1? function were studied in human dermal fibroblasts, SKRC7 cells and HIF-1? knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, Quantitative-PCR and Luciferase hypoxia-responsive element (HRE) reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1?-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1? at several levels by increasing both HIF-1? stability (by a non-canonical mechanism) and activity (as show both by induction of relevant target-genes and by a specific reporter assay). HIF-1? induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1? is knocked down. Moreover, the local transfer of stable HIF-1?-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) has an additive effect on HBO-mediated improvements in wound healing. In conclusion HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.
View details for DOI 10.1111/wrr.12253
View details for PubMedID 25532619
Selective blockade of estrogen receptor beta improves wound healing in diabetes.
Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes
2012; 43 (1): 127-134
Hypoxia plays a major pathogenic role in diabetic nephropathy (DN). We have investigated in this study the effect of hypoxia-inducible factor 1 ? subunit (HIF1A) genetic polymorphisms on the development of DN.In 1,165 American type 1 diabetic patients with and without DN selected from the Genetics of Kidneys in Diabetes (GoKinD) study, the HIF1A genetic polymorphisms were genotyped with TaqMan allelic discrimination. The regulation of HIF-1? in the kidneys of diabetic mice was appreciated by immunohistochemistry, and the effect HIF1A Pro582Ser polymorphism on HIF-1? sensitivity to glucose was evaluated in vitro.We identified a protective association between HIF1A Pro582Ser polymorphism and DN in male subjects. We also provided mechanistic insights that HIF-1? is repressed in the medulla of diabetic mice despite hypoxia and that Pro582Ser polymorphism confers less sensitivity to the inhibitory effect of glucose during a hypoxic challenge.The current study demonstrates for the first time that HIF1A Pro582Ser polymorphism has an effect on DN, possibly by conferring a relative resistance to the repressive effect of glucose on HIF-1?.
View details for DOI 10.2337/dc12-1125
View details for Web of Science ID 000314467100046
View details for PubMedID 22991450
Acute hypoxia induces apoptosis of pancreatic ß-cell by activation of the unfolded protein response and upregulation of CHOP.
Cell death & disease
Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100 mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.
View details for DOI 10.1007/s00726-012-1269-z
View details for Web of Science ID 000305210800014
View details for PubMedID 22451275
Stability of mitochondrial DNA against reactive oxygen species (ROS) generated in diabetes
DIABETES-METABOLISM RESEARCH AND REVIEWS
2011; 27 (5): 470-479
The success of pancreatic ?-cells transplantation to treat type 1 diabetes has been hindered by massive ?-cell dysfunction and loss of ?-cells that follows the procedure. Hypoxia-mediated cell death has been considered one of the main difficulties that must be overcome for transplantation to be regarded as a reliable therapy. Here we have investigated the mechanisms underlying ?-cell death in response to hypoxia (1% O(2)). Our studies show that mouse insulinoma cell line 6 (Min6) cells undergo apoptosis with caspase-3 activation occurring as early as 2?h following exposure to hypoxia. Hypoxia induces endoplasmic reticulum stress in Min6 cells leading to activation of the three branches of the unfolded protein response pathway. In response to hypoxia the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) is upregulated. The important role of CHOP in the apoptotic process was highlighted by the rescue of Min6 cells from hypoxia-mediated apoptosis observed in CHOP-knockdown cells. Culturing isolated pancreatic mouse islets at normoxia showed intracellular hypoxia with accumulation of hypoxia-inducible factor-1? and upregulation of CHOP, the latter one occurring as early as 4?h after isolation. Finally, we observed that pancreatic islets of type 2 db/db diabetic mice were more hypoxic than their counterpart in normoglycemic animals. This finding indicates that hypoxia-mediated apoptosis may occur in type 2 diabetes.
View details for DOI 10.1038/cddis.2012.66
View details for PubMedID 22695615
Epigenetic DNA methylation in the promoters of the Igf1 receptor and insulin receptor genes in db/db mice
2011; 6 (4): 405-409
Increased production of reactive oxygen species (ROS) in mitochondria has been proposed as the pathogenic mechanism for chronic complications of diabetes. Mitochondrial DNA (mtDNA) is more vulnerable to reactive oxygen species. However, there are few data on the mitochondrial DNA damage in diabetes and these are available only from patients with different duration of the disease and tissues not relevant to the chronic complications of diabetes. We therefore proposed to study the stability of mitochondrial DNA under controlled experimental conditions, to understand its contribution to chronic complications of diabetes.The mitochondrial DNA damage was evaluated by long-fragment polymerase chain reaction in human dermal fibroblasts exposed to high glucose level and hypoxia (an additional source of reactive oxygen species) or in organs from diabetic animals (db/db mice) at different ages. Reactive oxygen species production was assessed in vitro by fluorescence and in vivo by nitrosylation of the proteins. The antioxidant enzymes were assessed by enzyme activity and by quantitative real-time polymerase chain reaction while the mitochondrial repair activity (base excision repair) was determined by using abasic site-containing oligonucleotides as substrates.Hyperglycaemia, when combined with hypoxia, is able to induce mitochondrial DNA damage in human dermal fibroblasts. The deleterious effect is mediated by mitochondrial reactive oxygen species, being abolished when the mitochondria electron transport is blocked. The accumulation of mitochondrial DNA damage in vivo is, however, decreased in 'old' diabetic animals (db/db) despite higher reactive oxygen species levels. This mitochondrial DNA protection might be conferred by an increased base excision repair activity.Increased base excision repair activity in tissues affected by the chronic complications of diabetes is a potential mechanism that can overcome mitochondrial DNA damage induced by hyperglycaemia-related reactive oxygen species overproduction.
View details for DOI 10.1002/dmrr.1203
View details for Web of Science ID 000292772900007
View details for PubMedID 21484980
Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation
ELECTROMAGNETIC BIOLOGY AND MEDICINE
2011; 30 (2): 80-85
We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.
View details for Web of Science ID 000289066600002
View details for PubMedID 21474992
Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma
2010; 29 (30): 4276-4286
The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [(3)H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.
View details for DOI 10.3109/15368378.2011.566774
View details for Web of Science ID 000290733400003
View details for PubMedID 21591892
Stabilization of HIF-1 alpha is critical to improve wound healing in diabetic mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (49): 19426-19431
The Notch ligand delta-like ligand 4 (DLL4) is an essential component expressed by endothelial tip cells during angiogenic sprouting. We have described a conceptually novel therapeutic strategy for targeting tumor angiogenesis and endothelial tip cells based on DNA vaccination against DLL4. Immunization with DLL4-encoding plasmid DNA by in vivo electroporation severely retarded the growth of orthotopically implanted mammary carcinomas in mice by induction of a nonproductive angiogenic response. Mechanistically, vaccination brought about a break in tolerance against the self-antigen, DLL4, as evidenced by the production of inhibitory and inherently therapeutic antibodies against mouse DLL4. Importantly, no evidence for a delayed wound healing response, or for toxicity associated with pharmacological blockade of DLL4 signaling, was noted in mice immunized with the DLL4 vaccine. We have thus developed a well-tolerated DNA vaccination strategy targeting the endothelial tip cells and the antigen DLL4 with proven therapeutic efficacy in mouse models of mammary carcinoma; a disease that has been reported to dramatically induce the expression of DLL4. Conceivably, induction of immunity toward principal mediators of pathological angiogenesis could provide protection against recurrent malignant disease in the adjuvant setting.
View details for DOI 10.1038/onc.2010.176
View details for Web of Science ID 000280547900004
View details for PubMedID 20498640
The anti-papillomavirus activity of human and bovine lactoferricin
2007; 75 (3): 258-265
Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1alpha is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1alpha stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1alpha stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1alpha hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1alpha is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1alpha is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1alpha is critical to reverse the pathological process.
View details for DOI 10.1073/pnas.0805230105
View details for Web of Science ID 000261706600072
View details for PubMedID 19057015
Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17-31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1-49 showed some antiviral activity against HPV PsV infection while bLfcin 17-42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17-42 and hLfcin 1-49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.
View details for DOI 10.1016/j.antiviral.2007.03.012
View details for Web of Science ID 000249224600010
View details for PubMedID 17481742