Response to Comment on "Circadian rhythms in the absence of the clock gene Bmal1".

Ness-Cohn et al claim that our observations of transcriptional circadian rhythms in the absence of the core clock gene Bmal1 in mouse skin fibroblast cells are supported by inadequate evidence. They claim that they were unable to reproduce some of the original findings with their reanalysis. We disagree with their analyses and outlook.

Response to Comment on "Circadian rhythms in the absence of the clock gene Bmal1".

Abruzzi et al argue that transcriptome oscillations found in our study in the absence of Bmal1 are of low amplitude, statistical significance, and consistency. However, their conclusions rely solely on a different statistical algorithm than we used. We provide statistical measures and additional analyses showing that our original analyses and observations are accurate. Further, we highlight independent lines of evidence indicating Bmal1-independent 24-hour molecular oscillations.

Circadian rhythms in the absence of the clock gene Bmal1.

Circadian (~24 hour) clocks have a fundamental role in regulating daily physiology. The transcription factor BMAL1 is a principal driver of a molecular clock in mammals. Bmal1 deletion abolishes 24-hour activity patterning, one measure of clock output. We determined whether Bmal1 function is necessary for daily molecular oscillations in skin fibroblasts and liver slices. Unexpectedly, in Bmal1 knockout mice, both tissues exhibited 24-hour oscillations of the transcriptome, proteome, and phosphoproteome over 2 to 3 days in the absence of any exogenous drivers such as daily light or temperature cycles. This demonstrates a competent 24-hour molecular pacemaker in Bmal1 knockouts. We suggest that such oscillations might be underpinned by transcriptional regulation by the recruitment of ETS family transcription factors, and nontranscriptionally by co-opting redox oscillations.

Encapsulation of macrophages enhances their retention and angiogenic potential.

Cell therapies to treat critical limb ischaemia have demonstrated only modest results in clinical trials, and this has been partly attributed to poor cell retention following their delivery directly into the ischaemic limb. The aim of this study was to determine whether alginate encapsulation of therapeutic pro-angio/arteriogenic macrophages enhances their retention and ultimately improves limb perfusion. A reproducible GMP-compliant method for generating 300 µm alginate capsules was developed to encapsulate pro-angio/arteriogenic macrophages. Longitudinal analysis revealed no detrimental effect of encapsulation on cell number or viability in vitro, and macrophages retained their pro-angio/arteriogenic phenotype. Intramuscular delivery of encapsulated macrophages into the murine ischaemic hindlimb demonstrated increased cell retention compared with injection of naked cells (P = 0.0001), and that this was associated both enhanced angiogenesis (P = 0.02) and arteriogenesis (P = 0.03), and an overall improvement in limb perfusion (P = 0.0001). Alginate encapsulation of pro-angio/arteriogenic macrophages enhances cell retention and subsequent limb reperfusion in vivo. Encapsulation may therefore represent a means of improving the efficacy of cell-based therapies currently under investigation for the treatment of limb ischaemia.

The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy.

Anti-VEGF-A therapy has become a mainstay of treatment for ocular neovascularisation and in cancer; however, their effectiveness is not universal, in some cases only benefiting a minority of patients. Anti-VEGF-A therapies bind and block both pro-angiogenic VEGF-Axxx and the partial agonist VEGF-Axxxb isoforms, but their anti-angiogenic benefit only comes about from targeting the pro-angiogenic isoforms. Therefore, antibodies that exclusively target the pro-angiogenic isoforms may be more effective. To determine whether C-terminal-targeted antibodies could inhibit angiogenesis, we generated a polyclonal antibody to the last nine amino acids of VEGF-A165 and tested it in vitro and in vivo. The exon8a polyclonal antibody (Exon8apab) did not bind VEGF-A165b even at greater than 100-fold excess concentration, and dose dependently inhibited VEGF-A165 induced endothelial migration in vitro at concentrations similar to the VEGF-A antibody fragment ranibizumab. Exon8apab can inhibit tumour growth of LS174t cells implanted in vivo and blood vessel growth in the eye in models of age-related macular degeneration, with equal efficacy to non-selective anti-VEGF-A antibodies. It also showed that it was the VEGF-Axxx levels specifically that were upregulated in plasma from patients with proliferative diabetic retinopathy. These results suggest that VEGF-A165-specific antibodies can be therapeutically useful.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.

In vivo effects of tailored laminin-332 α3 conjugated scaffolds enhances wound healing: a histomorphometric analysis.

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration.

Patient-related keloid scar assessment and outcome measures.

BACKGROUND: Keloid scars cause pain, itching, functional limitation, and disfigurement, leading to psychological distress. Progress in treatment regimens is hindered by the lack of a universally accepted outcome measure. The Patient and Observer Scar Assessment Scale is a tool for the assessment of scars, incorporating an assessment by both clinician and patient. This study evaluates its application to keloids and compares it to the widely used Vancouver Scar Scale, which is considered the standard mode of assessment for scars. METHODS: Three observers using the two scales assessed 34 patients with 41 keloid scars independently. Patients evaluated their own scars simultaneously using the patient component of the Patient and Observer Scar Assessment Scale. Internal consistency, interobserver reliability, and convergent validity were examined. RESULTS: Both components of the Patient and Observer Scar Assessment Scale had high internal consistency (0.82 and 0.86 for patient and observer components, respectively); those rates were higher than the rate for the Vancouver Scar Scale (0.65). Interobserver reliability was "substantial" for the Vancouver Scar Scale (0.65) and "almost perfect" for the observer component of the Patient and Observer Scar Assessment Scale (0.85). Convergent validity was very strong (0.83, p < 0.01), although the patient component did not correlate well with either of the observer scales. Patients rated their scars worse than the observer average for 83 percent of the scars, and were influenced by color, stiffness, thickness, and irregularity (p < 0.05). CONCLUSION: The findings support the use of the Patient and Observer Scar Assessment Scale as a reliable and valid method of assessing keloid scars in a clinical context. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, II.

The effect of laminin peptide gradient in enzymatically cross-linked collagen scaffolds on neurite growth.

Guided neurite growth is critical in both peripheral nervous system and central nervous system nerve regeneration. Scaffolds that provide structural and guidance cues for neuronal cells have a potential role in neural regeneration application. Type I collagen is suitable to be processed as an engineered scaffold for nerve regeneration because of its biological and structural properties. A few previous studies have shown that cross-linking of collagen scaffolds with microbial transglutaminase improves the mechanical strength and degradation properties of the scaffolds. It was shown that laminin 5 can regulate neurite outgrowth and extension. A motif (PPFLMLLKGSTR) in the human laminin 5 alpha 3 chain is crucial for both integrin alpha 3 beta 1 receptor binding and cell adhesion. In the present work, we studied the guidance effect of a laminin peptide (PPFLMLLKGSTR) gradient in collagen and cross-linked collagen scaffolds on neurite growth. Neurites of rat pheochromocytoma (PC12) cells showed a preferential growth toward the high laminin concentration level on the collagen scaffold, while the incorporation of laminin peptide in the scaffold did not influence neurite length of PC12 cells.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion.

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality.

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Effect of functionalized micropatterned PLGA on guided neurite growth.

When coaptation is not possible in the repair of nerve injuries, a bridge of biomaterial scaffold provides a structural support for neuronal cell growth and guides nerve regeneration. Poly(lactide-co-glycolide) (PLGA) scaffolds have been widely investigated for neural tissue engineering applications. In order to investigate guided neurite growth, we have fabricated micropatterns on PLGA films using laser ablation methods. The micropatterned PLGA films were coated with collagen type I or laminin peptide (PPFLMLLKGSTR) to promote axon growth. Micropatterned PLGA films provide a guidance effect on both early stage neurite outgrowth and elongation. Small (5 microm) grooves showed more statistically significant parallel neurite growth compared with larger size grooves (10 microm). Micropatterned PLGA films coated with laminin peptide showed more parallel neurite growth compared with those coated with collagen type I. Primary neurite number and total neurite length per cell decreased on micropatterned PLGA films compared with the controls. Neurites showed a preference for growth in the microgrooves rather than on the spaces. This study indicates that surface micropatterned structures with conjugated functional molecules can be used to guide neurite growth.

Enhancing amine terminals in an amine-deprived collagen matrix.

Collagen, though widely used as a core biomaterial in many clinical applications, is often limited by its rapid degradability which prevents full exploitation of its potential in vivo. Polyamidoamine (PAMAM) dendrimer, a highly branched macromolecule, possesses versatile multiterminal amine surface groups that enable them to be tethered to collagen molecules and enhance their potential. In this study, we hypothesized that incorporation of PAMAM dendrimer in a collagen matrix through cross-linking will result in a durable, cross-linked collagen biomaterial with free -NH 2 groups available for further multi-biomolecular tethering. The aim of this study was to assess the physicochemical properties of a G1 PAMAM cross-linked collagen matrix and its cellular sustainability in vitro. Different amounts of G1 PAMAM dendrimer (5 or 10 mg) were integrated into bovine-derived collagen matrices through a cross-linking process, mediated by 5 or 25 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 5 mM N-hydroxysuccinimide (NHS) and 50 mM 2-morpholinoethane sulfonic acid buffer at pH 5.5. The physicochemical properties of resultant matrices were investigated with scanning electron microscopy (SEM), collagenase degradation assay, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectra, and ninhydrin assay. Cellular sustainability of the matrices was assessed with Alamar Blue assay and SEM. There was no significant difference in cellular behavior between the treated and nontreated groups. However, the benefit of incorporating PAMAM in the cross-linking reaction was limited when higher concentrations of either agent were used. These results confirm the hypothesis that PAMAM dendrimer can be incorporated in the collagen cross-linking process in order to modulate the properties of the resulting cross-linked collagen biomaterial with free -NH 2 groups available for multi-biomolecular tethering.