Kerry Keefer

Hyaluronan induces scarless repair in mouse limb organ culture

BACKGROUND/PURPOSE Wounded fetal mouse limbs harvested from two distinct time points in gestation heal differently in organ culture. The healing of a gestational day 14 limb is by scarless repair, whereas gestational day 18 (gd 18) limbs heal by scarring. The persistence of elevated levels of hyaluronic acid (HA) is a major difference in the extracellular matrix of scarless repair. The purpose of this study was to demonstrate that chronic additions of HA to incisional wounds of gd 18 limbs induces scarless repair. METHODS Time-dated pregnant CD-1 mice (term, 20 days) were killed on gestational day 18 and fetuses were harvested via laparotomy. A through and through stab wound was made in each forelimb with a 1-mm microscapel, and the wound was closed with a single 10-0 nylon suture. The forelimbs were amputated at the level of the shoulder and placed in organ culture. Daily medium changes with 1 mL of BGJb (devoid of serum) were made. Half the cultures received 10 microL of HA (4 mg/mL) directly to the wound site with each medium change. The other half of the cultures received 10 microL of phosphate-buffered saline (PBS-control). At day 7, the limbs were harvested, fixed in methyl Carnoys solution, paraffin embedded, and 5-microm serial sections cut. The sections were stained with H&E or Sirius red/fast green. The sections were viewed in a blinded fashion by two observers. Suture defined the wound site, and the sections were graded for healing by scarring. RESULTS Minimal limb growth occurred in both control and HA-treated limbs. Grossly, both control and treated limbs healed incisional wounds by 7 days in culture. Limbs from both treatment and control groups showed viability by microscopic analysis. The limbs treated with HA had no appreciable scar morphologically in sections in which epithelial dimpling and suture were evident. The orientation of the collagen fiber bundles in the control wounds were in parallel arrays perpendicular to the incision. The orientation of the collagen fiber bundles in the HA-treated limbs had a basket weave pattern that was indistinguishable from unwounded dermis. The direct repeated additions of HA to healing organ cultured limb explants of gestational day 18 fetal mice promoted scarless repair. CONCLUSIONS This result demonstrates that chronic elevation of HA in the microenvironment of a wound affects healing by promoting the deposition of a more dermal-like connective tissue matrix in the wound site. The maintenance of elevated levels of HA could have utility in the clinical setting to improve the organization of connective tissue, leading to the reduction of scar complications.

Repeated additions of hyaluronan alters granulation tissue deposition in sponge implants in mice

The role for the metabolism of hyaluronic acid in the repair process is uncertain. Fetal dermal wounds do not heal by scarring and have sustained high levels of hyaluronic acid. In contrast, adult dermis is repaired by scarring and has less hyaluronic acid. Initially after injury, hyaluronic acid is elevated in both adult and fetal wounds, and although it remains elevated in fetal repair, it is rapidly degraded in adult wounds. The chronic addition of hyaluronic acid or hyaluronidase to polyvinyl alcohol sponge implants in adult mice was investigated in this study. Polyvinyl alcohol sponge implants containing a central reservoir were placed subcutaneously in the dorsum of adult male CD‐1 mice. Mice were divided into three groups: a phosphate‐buffered saline control, a 20 µg hyaluronic acid treatment group, and a 10 U hyaluronidase treatment group. The central reservoir of each sponge implant received appropriate compound every 3 days for 2 weeks via transdermal injection and were then evaluated histologically. At 2 weeks, the cellular density and the quantity of granulation tissue deposition were the greatest in the hyaluronidase group and were lowest in the hyaluronic acid group. In addition, the organization of collagen fiber bundles was the most dense in the hyaluronidase group and least in the hyaluronic acid group. In a second experiment, polyvinyl alcohol sponge implants in mice received either phosphate‐buffered saline solution or 20 µg hyaluronic acid every 3 days for 1 week. On day 5, an aliquot of fluorescently tagged native collagen was injected into the sponges. Sponges were harvested at day 7, cryosections made, and the presence of autofluorescent collagen fibers assessed. The autofluorescent collagen fiber bundles in the phosphate‐buffered saline solution group were organized in thick parallel bundles, whereas the collagen bundles from hyaluronic acid‐treated implants were organized in fine lacelike structures. Chronic addition of hyaluronic acid appears to mimic the fetal dermal connective tissue matrix in which repair proceeds with diminished collagen deposition, organized in finer collagen fiber bundles in granulation tissue. On the other hand, the removal of hyaluronic acid by the chronic administration of hyaluronidase increases the amount of granulation tissue. Elevated levels of hyaluronic acid in granulation tissue appear to modulate the ability of resident fibroblasts to organize collagen fiber bundles.

Regional cerebral and neural lobe blood flow during insulin-induced hypoglycemia in unanesthetized rats

The effects of hypoglycemia on regional cerebral blood flow (rCBF) were studied in awake restrained rats. The rats were divided into three groups consisting of (1) a normoglycemic control group that received only saline, (2) a hypoglycemic group A, which was given insulin 30 min before flow was measured, and (3) a hypoglycemic group B, which was given insulin 90 and 30 min before flow was measured. Regional CBF was measured using 14C-iodoantipyrine. Mean plasma glucose was 8.76 μmol/ml in the control group, 2.63 μmol/ml in hypoglycemic group A, and 1.51 μmol/ml in hypoglycemic group B. Plasma epinephrine and norepinephrine concentrations increased to approximately 375% and 160%, respectively, of control values in hypoglycemic groups A and B. In the hypoglycemic group A, rCBF significantly increased in three brain regions. In the hypoglycemic group B, rCBF increased significantly in all brain regions measured, with the exception of the neural lobe, in which it decreased. The increase in rCBF ranged from 38% in the hypothalamus to 138% in the thalamus. Neural lobe blood flow significantly decreased by 31%. The neural lobe was the only brain region studied that is not protected by a blood-brain barrier. It may be sensitive to changes in the concentration of vasoactive agents in blood, such as epinephrine and norepinephrine.

Regional Cerebral Glucose Utilization During Insulin-Induced Hypoglycemia in Unanesthetized Rats

Regional cerebral glucose utilization (rCMRgl) was studied during insulin‐induced hypoglycemia in unanesthetized rats. Rats were surgically prepared using halothane and nitrous oxide anesthesia and allowed 5 h to recover from the anesthesia before rCMRgl was measured. The rCMRgl was measured using [6‐14C]glucose in a normoglycemic control group and two hypoglycemic groups, A (30 min after insulin injection) and B (2 h after insulin injection). The mean plasma glucose level was 7.03 μmol/ml in the normoglycemic group, 1.96 μmol/ml in hypoglycemic group A, and 1.40 μmol/ml in hypoglycemic group B. The rCMRgl in hypoglycemic group A decreased 8–18% in 17 brain regions measured; five changes were statistically significant. The rCMRgl in hypoglycemic group B decreased significantly in all but one of the brain regions measured; the decrease ranged from 15% in the pyramidal tract to 36% in the motor and auditory cortices. The rCMRgl in every brain region decreased when the plasma glucose level fell below 1.5–2.5 μmol/ml. No brain region could maintain rCMRgl at plasma glucose concentrations lower than predicted by regional glucose influx described in previous studies. Glucose utilization in all brain regions appears to be limited by the influx of glucose.

The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

A Splice Variant of the Human Ion Channel TRPM2 Modulates Neuroblastoma Tumor Growth through Hypoxia-inducible Factor (HIF)-1/2α

Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2α expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2α. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics. The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy.

Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing.

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.

Mouse skin chemical carcinogenesis is inhibited by antizyme in promotion-sensitive and promotion-resistant genetic backgrounds.

Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)‐ or K6‐driven AZ transgenes on 7,12‐dimethylbenz[a]anthracene (DMBA)/12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) chemical carcinogenesis of the skin, in promotion‐resistant C57BL/6 and promotion‐sensitive DBA/2 mice. On both genetic backgrounds, K6‐AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild‐type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5‐AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5‐AZ and K6‐AZ groups. Tumor and TPA‐treated skin sections from K6‐AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6‐AZ mice also had slightly reduced cell proliferation rates in tumors and TPA‐treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor‐suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin

Modulation of intracellular calcium ([Ca2+]i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normal human erythroid precursors, but Epo stimulates an increase in [Ca2+]i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca2+]i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.

The role of transforming growth factor‐β in the conversion from “scarless” healing to healing with scar formation

The objective of this study was to elucidate mediators responsible for conversion of “scarless” wound healing seen in wounded, day 14 fetal mouse limbs to healing with scar formation seen in wounded, day 18 fetal mouse limbs. Wounded, day 14 limbs were grown in a serum‐free organ culture system in which either phosphate‐buffered saline solution or human recombinant transforming growth factor beta‐1 (1 µg/ml) was added daily. Wounded, day 18 limbs were also maintained in the same organ culture system with either phosphate‐buffered saline solution or neutralizing antibody to transforming growth factor‐β (1 µg/ml) treatment. Limb cross sections were examined qualitatively with Massons Trichrome stain and quantitatively by spectrophotometric analysis of Sirius Red and Fast Green dyes which bind to collagen and noncollagenous protein, respectively. Both qualitative and quantitative analyses showed the following: there was greater collagen deposition in day 18 versus day 14 limbs by 7 days after wounding, scar formation in day 18 limbs was attenuated by the addition of anti‐transforming growth factor‐β, and there was the addition of transforming growth factor‐β‐augmented collagenous scar formation in wounded regions of day 14 limbs. These results strongly suggest that transforming growth factor‐β present in the local wound environment is, at least in part, responsible for the conversion of “scarless” healing occurring in wounded, day 14 limbs to scar formation present in wounded, day 18 limbs.