Unravelling novel molecular targets for photobiomodulation in human hair follicle towards the development of more effective light-based therapies for hair growth

Light and optical techniques have made a profound impact on modern medicine both in diagnostics and in therapy. Therapeutic action of light is based on photomechanical, photothermal, photochemical and photobiological interactions, depending on the wavelength, power density, exposure time and optical properties of tissue and cells. Last decade experienced a growing rise of commercial devices for management of hair growth, where all of them are based on low levels of light resulting into photobiological, non-thermal interaction of photons with cells, a process that recently has received an official term ‘photobiomodulation’. However, the design and analysis of the reported clinical studies are highly debated in a wider scientific community. The picture is further complicated by a virtual lack of proof about the exact molecular targets that mediate the physiological response of skin and hair follicles (HF) to low levels of light. The goal of this project was to investigate the expression of light-sensitive receptors in the human HF and to study the impact of UV-free blue light on hair growth ex vivo. The expression of Cryptochromes 1 and 2 (CRY1, 2), Opsin 2 and 3 (OPN2 and OPN3), but not other Opsins 1, 4 and 5 was detected in the distinct compartments of skin and anagen HF. Evaluation of the physiological role of detected light-sensitive receptors on hair growth was performed by the modulation of photoreceptors activity in HF ex vivo model. HFs treated with KL001, a stabilizer of CRY1 protein that lengthens the circadian period, delayed HF anagen-catagen transition; while silencing of CRY1 induced premature catagen development accompanied by reduced cell proliferation. Silencing of CRY1 in the HF outer root sheath (ORS) cells in vitro caused downregulation of ii genes involved in the control of proliferation; including the cyclin dependent kinase 6 (CDK6). OPN3 also had a positive effect on metabolic activity and proliferation of the ORS cells in vitro. OPN3 silencing resulted in the altered expression of genes involved in the control of proliferation and apoptosis. Investigated CRY1, OPN2 and 3 greatly absorb in the blue to green-region of the visible spectrum. This led us to investigate the effect of blue light on HF growth. Daily treatment with blue light (453 nm, 3.2 J/cm2, 16 nm full width half maximum) prolonged anagen phase in HF ex vivo that was associated with sustained proliferation. In addition, blue light (3.2 J/cm2) significantly stimulated proliferation of ORS cells in vitro. This effect was abrogated by silencing of OPN3. To summarize, CRY 1, OPN 2 and OPN 3 are expressed in the distinct compartments of the HF, including HF stem cells. Blue light (453 nm) at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. The further research should be conducted to decipher interactions between blue light and the investigated receptors in the HFs. In addition, the beneficial effect of blue light at low radiant exposure on hair growth raises a possibility of increasing therapeutic efficacy when combined with topical chemistry used for management of hair growth

Cryptochrome 1 is modulated by blue light in human keratinocytes and exerts positive impact on human hair growth

Photoactivation of cryptochrome-family proteins by blue light is a well-established reaction regulating physiology of plants, fungi, bacteria, insects and birds, while impact of blue light on cryptochrome synthesis and/or activity in human non-visual cells remains unknown. Here, we show that 453 nm blue light induces cryptochrome 1 (CRY1) accumulation in human keratinocytes and the hair follicle. CRY1 is prominently expressed in the human anagen hair follicle, including epithelial stem cells. Specific silencing of CRY1 promotes catagen, while stimulation of CRY1 by KL001 prolongs anagen ex vivo by altering the expression of genes involved in apoptosis and proliferation. Together, our study identifies a role for CRY1 in sustaining human hair growth. Previously, we demonstrated positive effects of 453 nm blue light on hair growth ex vivo. Taken all together, our study suggests that CRY1 might mediate blue light-dependent positive effects on hair growth

Phenotype and distribution of immature T cells in adenoids and tonsils.

<p>A: macroscopic appearance of a representative sample of tonsils and adenoidal tissue. B: Low power (4x) image of a fixed and embedded adenoid, stained with Hematoxylin and Eosin. The brackets highlights the boundary between the stroma and the secondary lymphoid tissue (star  =  germinal center) where darker staining small lymphocytes gather. Left lower corner inset shows TdT+(brown) cells in sheets in an immunostained serial section (4x). Upper right inset: H&E detail of the bracketed area (40x). C-H: frozen adenoid serial sections (4x) stained for CD1a (C), CD1b (D), TE-7 (E), NGFR/p75 (F), and the anti thymic epithelium reagents RFD4 (G) and TE-4 (H) (brown, no counterstain). The insets in C, E and F show a magnified detail of the area occupied by immature T cells (40x)(also indicated by brackets). Star  =  germinal center. The insets in G and H show low power images (4x). The arrows highlight positive surface epithelium. I: frozen adenoid serial sections (40x) double stained for negative controls, TdT and CD1a, CD4, CD8, CD79a and surrogate light chain (SL; inset). Note the doublestaining in the upper half field, except for CD79a and SL. The last image is a double immunofluorescence image showing largely mutually exclusive distribution of Pax5 and TdT in adenoids in a fixed and embedded section. K: fixed and embedded tonsil section showing coexpression of TdT and Pax5 in immature B cell precursors.</p

Distinct Effects of Heparin and Interleukin-4 Functionalization on Macrophage Polarization and In Situ Arterial Tissue Regeneration Using Resorbable Supramolecular Vascular Grafts in Rats

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months). Routine analyses are augmented with Raman microspectroscopy, in order to acquire the local molecular fingerprints of the resorbing scaffold and developing tissue. Thrombosis is found not to be a confounding factor in any of the groups. Hep-only-functionalized grafts resulted in adverse tissue remodeling, with cases of local intimal hyperplasia. This is negated with the addition of IL-4, which promoted M2 macrophage polarization and more mature neotissue formation. This study shows that with bioactive functionalization, the early inflammatory response can be modulated and affect the composition of neotissue. Nevertheless, variability between graft outcomes is observed within each group, warranting further evaluation in light of clinical translation

Ultrastructural Characteristics of Myocardial Reperfusion Injury and Effect of Selective Intracoronary Hypothermia: An Observational Study in Isolated Beating Porcine Hearts

In acute myocardial infarction (AMI), myocardial reperfusion injury may undo part of the recovery after revascularization of the occluded coronary artery. Selective intracoronary hypothermia is a novel method aimed at reducing myocardial reperfusion injury, but its presumed protective effects in AMI still await further elucidation. This proof-of-concept study assesses the potential protective effects of selective intracoronary hypothermia in an ex-vivo, isolated beating heart model of AMI. In four isolated Langendorff perfused beating pig hearts, an anterior wall myocardial infarction was created by inflating a balloon in the mid segment of the left anterior descending (LAD) artery. After one hour, two hearts were treated with selective intracoronary hypothermia followed by normal reperfusion (cooled hearts). In the other two hearts, the balloon was deflated after one hour, allowing normal reperfusion (control hearts). Biopsies for histologic and electron microscopic evaluation were taken from the myocardium at risk at different time points: before occlusion (t = BO); 5 minutes before reperfusion (t = BR); and 10 minutes after reperfusion (t = AR). Electron microscopic analysis was performed to evaluate the condition of the mitochondria. Histological analyses included evaluation of sarcomeric collapse and intramyocardial hematoma. Electron microscopic analysis revealed intact mitochondria in the hypothermia treated hearts compared to the control hearts where mitochondria were more frequently damaged. No differences in the prespecified histological parameters were observed between cooled and control hearts at t = AR. In the isolated beating porcine heart model of AMI, reperfusion was associated with additional myocardial injury beyond ischemic injury. Selective intracoronary hypothermia preserved mitochondrial integrity compared to nontreated controls