Global periorbital skin rejuvenation by a topical eye cream containing low molecular weight heparan sulfate (LMW-HS) and a blend of naturally derived extracts.

BACKGROUND: Maintaining a youthful appearance is a priority for many people. Global eye rejuvenation is sought more frequently and at a younger age than other treatments. Major concerns around the eye area are periorbital hyperpigmentation, puffiness, and lines and wrinkles. Glycosaminoglycans (GAGs) are complex carbohydrates that modulate skin health, repair and renew skin's appearance. Heparan sulfate (HS) is the most biologically active GAG, although it is too large and polar to penetrate the skin. Low Molecular Weight Heparan Sulfate (LMW-HS) is a smaller version of HS designed for skin penetration while preserving its activity. In this study, we investigated the effects of a topical eye cream containing LMW-HS and a blend of naturally derived extracts to address global periorbital rejuvenation. METHOD: A single-center, open-label study including female and male subjects (n = 15) was conducted to evaluate the efficacy and tolerability of an eye cream containing LMW-HS and a blend of naturally derived extracts applied twice daily for 12 weeks. RESULTS: Improvements in the appearance of periorbital hyperpigmentation and fine and coarse wrinkles were observed as early as week 2 with continuous improvement up to 12 weeks. Decrease in puffiness (73%) and dark circles (93%) were reported by subjects. The test product was highly rated by subjects on performance and attributes and was well tolerated by all the subjects in this study. CONCLUSION: Results demonstrated that an eye cream containing LMW-HS and a blend of naturally derived extracts achieved global skin rejuvenation by improving appearance of periorbital hyperpigmentation, puffiness, and fine and coarse wrinkles.

Rejuvenating Hydrator: Restoring Epidermal Hyaluronic Acid Homeostasis With Instant Benefits.

Skin aging is a combination of multifactorial mechanisms that are not fully understood. Intrinsic and extrinsic factors modulate skin aging, activating distinctive processes that share similar molecular pathways. One of the main characteristics of youthful skin is its large capacity to retain water, and this decreases significantly as we age. A key molecule involved in maintaining skin hydration is hyaluronic acid (HA). Concentration of HA in the skin is determined by the complex balance between its synthesis, deposition, association with cellular structures, and degradation. HA bio-equivalency and bio-compatibility have been fundamental in keeping this macromolecule as the favorite of the skincare industry for decades. Scientific evidence now shows that topically applied HA is unable to penetrate the skin and is rapidly degraded on the skin surface. SkinMedica's HA5 Rejuvenating Hydrator (SkinMedica Inc., an Allergan company, Irvine, CA) promotes restoration of endogenous epidermal HA homeostasis and provides instant smoothing and hydration of the skin. These dual benefits are accomplished through the combination of 2 breakthrough technologies: 1) a unique blend of actives powered by SkinMedica proprietary flower-derived stem cell extract that restores the endogenous production of HA; and 2) a proprietary mix of 5 HA forms that plump the skin, decreasing the appearance of fine lines/wrinkles. Pre-clinical studies demonstrated that HA5 induces expression of key epidermal differentiation and barrier markers as well as epidermal HA synthases. A decrease expression of hyaluronidases was also observed upon HA5 application. Initial clinical studies showed that within 15 minutes of application, HA5 instantly improves the appearance of fine lines/wrinkles and skin hydration. Subjects that continue using HA5 (for 8 weeks) demonstrated significant improvements in fine lines/wrinkles, tactile roughness, and skin hydration. In summary, the blend of these 2 key technologies present in HA5 promotes restoration of endogenous epidermal HA while delivering instant smoothing effects.

Total Defense + Repair: A Novel Concept in Solar Protection and Skin Rejuvenation.

For more than a century, solar radiation has been known to contribute significantly to the extrinsic aging of skin. Until recently, this was almost exclusively attributed to the photodamage caused by ultraviolet (UV) light. However, a growing body of evidence now indicates that both infrared (IR) and visible light may also contribute to extrinsic skin aging. Infrared radiation, comprised of IR-A, IR-B, and IR-C, accounts for 54.3% of the total solar radiation reaching the skin. Studies have shown that IR radiation is also responsible for skin aging. Thus, IR-A radiation regulates hundreds of genes in skin, with roles in extracellular matrix (ECM) homeostasis regulation, apoptosis, cell growth, and stress responses. IR-B and IR-C radiation are primarily responsible for the increase in skin temperature associated with solar exposure, and are implicated in heat-related skin destruction of collagen and elastin, which is characterized by an increase in the expression of matrix metalloproteinases (MMPs). The contribution of visible light to photoaging is less well understood; however, some preliminary indication associates visible light with the upregulation of MMPs' expression, DNA damage, and keratinocyte proliferation. Interestingly, the common denominator that links skin damage to the different solar wavelengths is the enhanced production of reactive molecule species (RMS) and therewith increased oxidative stress. SkinMedica® Total Defense + Repair (TD+R; SkinMedica Inc., an Allergan company, Irvine, CA) is a "superscreen," which combines broad spectrum UV protection with a unique blend of antioxidants (SOL-IR Advanced Antioxidant Complex™) that provide protection from IR radiation while promoting skin repair. Preclinical studies have indicated that TD+R SPF34 prevents the formation of UV-induced sunburn cells and cyclobutane pyrimidine dimers while preserving or improving the expression of ECM genes. In addition, it prevents IR-A-triggered fragmentation of elastin fibers and expression of MMP-1. Initial clinical studies indicate that TDR+R SPF34 reduces the increase in surface temperature seen with IR radiation. A significant improvement in the appearance of lines and wrinkles was reported as early as week 2 in patients using TDR+R SPF34. In summary, we observed that the unique blend of antioxidants present in TD+R acts in harmony with SPF active ingredients, expanding solar protection beyond UV radiation and counterbalancing the deleterious effects of free radicals on skin cells by promoting endogenous repair.

Activation of the stress response in macrophages alters the M1/M2 balance by enhancing bacterial killing and IL-10 expression.

UNLABELLED: Macrophages (Mϕs) play an important role in the inflammatory response during injury by participating in the removal of injurious stimuli, such as bacteria, and promoting tissue healing to restore homeostasis. Mϕs can acquire distinct functional phenotypes along a spectrum between two opposite stages (M1/M2) during activation. In the present study, we induced a stress response in Mϕs via heat shock (HS) and found that it incurred an increase in phagocytosis (1.6-fold, P < 0.05) and bacterial killing (2.8-fold, P < 0.01). Upon heat stress activation, Mϕs respond to group B Streptococcus (GBS) infection with lower levels of pro-inflammatory cytokines, TNF-α (2.25-fold, P < 0.01), IL-6 (7-fold, P < 0.001), and inducible nitric oxide synthase (iNOS) (2.22-fold, P < 0.05), but higher levels of the anti-inflammatory cytokine IL-10 (3.9-fold, P < 0.01). Stressed Mϕs exposed to GBS display rapid phagosome maturation, increased extracellular trap (ET) formation and elevated cathelicidin antimicrobial peptide expression (2.5-fold, P < 0.001). These findings are consistent with a heretofore uncharacterized Mϕ activation state formed in response to stress, associated with secretion of large quantities of anti-inflammatory mediators and redirection of antimicrobial mechanisms to NADPH-oxidase-independent pathways. This "friendly activation" of Mϕs is characterized by increased bactericidal activity and more rapid and controlled resolution of the inflammatory response. KEY MESSAGES: Macrophages form a dual pro-bactericidal and anti-inflammatory state. Stress in the setting of infection triggers friendly activation in macrophages. Heat shock plus infection increases macrophage bactericidal activity. Heat shock plus infection increases macrophage extracellular trap formation. Heat shock plus infection increases macrophage production of cathelicidin and IL-10.

Tumor necrosis factor expression is ameliorated after exposure to an acidic environment.

BACKGROUND: It has been well established that laparoscopic surgery presents several clinical benefits, including reduced pain and a shorter hospital stay. These effects have been associated with a decrease in the inflammatory response. Previous studies have demonstrated that reduced inflammation after laparoscopic surgery is the product of carbon dioxide insufflation, which decreases peritoneal pH. The objective of this study was to investigate the cellular and molecular mechanisms responsible for the reduced response after exposure to acidic environments. MATERIALS AND METHODS: A murine macrophage line (J744) was incubated in culture medium at pH 6.0 or pH 7.4 for 3 h at 37°C. Then, cells were stimulated with lipopolysaccharide (LPS) at pH 7.4, the expression of TNF-α (qRT-PCR or enzyme-linked immunosorbent assay (ELISA) and intracellular pH were measured. In addition, CD14 and Toll-like receptor 4 expression and NF-κB nuclear translocation were analyzed. RESULTS: A significant decrease in LPS-induced TNF-α expression levels was observed in cells pre-incubated at pH 6.0 in comparison with cells at neutral pH conditions. This decrease in TNF-α levels was not associated with a reduction in cell surface expression of CD14 and Toll-like receptor 4. Exposure to an extracellular acidic environment resulted in a reduction of IκB phosphorylation and NF-κB nuclear translocation, secondary to a significant drop in cytosolic pH. CONCLUSIONS: These observations provide a potential mechanism for the reduced expression of TNF-α after exposure to low extracellular pH, which may be related to acidification after CO(2) insufflation during laparoscopic surgery. In addition, extracellular acidic pH environments could emerge as an important regulator of macrophage function.

A new feature of the stress response: increase in endocytosis mediated by Hsp70.

The expression of heat shock proteins (HSP) is a conserved cellular response to a variety of stresses. These proteins have been found to refold denatured polypeptides and stabilize critical cellular processes. In this study, we introduce a new component of the stress response: the increase of receptor-mediated uptake of macromolecules from the external environment. We observed that endocytosis of transferrin, which is involved in the delivery of iron to the cell, was increased after stress induced by heat shock or after incubation with inhibitors of Hsp90 function. In both cases, the increase in endocytosis was reverted by inhibition of transcription, suggesting that gene expression is required. Transfection of cells with Hsp70 gene or inhibition of its expression by siRNA confirmed the role of this HSP in the increase of endocytosis. The mechanism for the enhancement of transferrin uptake was related to an accelerated internalization of the ligand-receptor complex as well as an increase in receptor recycling. These observations constitute a new paradigm for the cellular protection induced by HSP.

Acidification enhances peritoneal macrophage phagocytic activity.

BACKGROUND: Laparoscopic surgery is currently used in an array of diverse clinical situations, including cases with potential bacterial contamination. Previous studies have shown that CO2 insufflation during laparoscopic procedures modulates the immune response due to the acidification of the peritoneum. In the present study, we investigated whether exposure of macrophages to an acidic environment, such as that produced by CO2 insufflation, could affect phagocytosis, which is the fundamental process for bacterial clearance. MATERIALS AND METHODS: A murine peritoneal macrophage line (J774) was pre-incubated at pH levels of 6.0 or 7.4 for 3 h at 37 degrees C and returned to neutral pH (7.4). Phagocytosis was evaluated by incubation with fluorescein isothiocyanate-conjugated IgG-opsonized bacterial particles, IgG-opsonized fluorescent latex beads, and non-opsonized fluorescent latex beads at 37 degrees C, pH 7.4. The intensity of the internalized signal was measured by using a fluorometer. RESULTS: Pre-incubation of macrophages at a pH of 6.0 resulted in a significant increase of phagocytic activity of opsonized particles. However, it did not change the uptake of non-opsonized particles. This effect was due to the internalization process since there were no differences in foreign particle binding of cells exposed to acidic or neutral pH levels. CONCLUSIONS: This study demonstrates that environmental acidification increases the phagocytosis of opsonized particles by macrophages. These results suggest that CO2 insufflation during laparoscopic surgery may be beneficial for the clearance of pathogens, particularly in cases where there is a high risk of potential intra-abdominal infections.

Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages.

Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.

Increase in phagocytosis after geldanamycin treatment or heat shock: role of heat shock proteins.

The response to injury is activated at the systemic and cellular levels. At the systemic level, phagocytosis plays a key role in controlling infections and clearing necrotic and apoptotic cells. The expression of heat shock proteins (Hsp), which is a well-conserved process, is a major component of cellular response to stress. This study investigated the relationship between Hsps and phagocytosis. An increase in the phagocytosis of opsonized bacteria particles and latex beads was observed upon incubation of murine macrophages with geldanamycin (GA), a specific inhibitor of the Hsp90 family of proteins. The effect of GA on phagocytosis was blocked by coincubation with inhibitors of transcription (actinomycin D) or translation (cycloheximide), suggesting that gene expression was required. Because expression of Hsps has been observed after GA treatment, the effect of heat shock on phagocytosis was investigated. Similar to GA treatment, heat shock resulted in an actinomycin D-sensitive elevation of phagocytosis, which suggests that Hsps are involved. The increase in phagocytosis after GA treatment was not due to increased binding of opsonized particles to their respective receptors on the macrophage surface or to elevated oxidative stress. However, it was correlated with a rapid polymerization of actin in proximity to the plasma membrane. These results suggest that Hsps play a role in the modulation of the phagocytic process, which is part of the stress response.

IL-10 plasma levels are elevated after LPS injection in splenectomized A/J mice.

BACKGROUND: Splenectomy is clinically indicated in certain cases of hypersplenism and splenic trauma. However, it is associated with serious complications, in particular, reduced clearance of encapsulated organisms and a high incidence of sepsis, which has been coined overwhelming post-splenectomy sepsis (OPSS). In addition to the role of the spleen in the clearance of microorganisms, this organ may be involved in regulation of the inflammatory response. We investigated the effect of splenectomy on the inflammatory process induced by LPS in a murine model that resembles, in part, the pathophysiological aspects of sepsis. MATERIALS AND METHODS: Male mice (8-weeks-old) from different inbred strains were randomized into three groups: splenectomized (SPX), sham operated (SHAM), and non-operated controls (NoOp). After 9 days of recovery, mice were injected with LPS (15 mg/kg) and cytokine plasma levels were measured by ELISA at 1.5 or 6 h after injection. Peritoneal macrophages (PMphi) were isolated from the three groups, and cytokine production was evaluated after incubation with LPS in culture conditions. RESULTS: IL-10 plasma levels were elevated in SPX A/J mice (6.7 +/- 0.4 mug/ml) after injection of LPS (15 mg/kg) compared to NoOp A/J mice (4.2 +/- 0.2 mug/ml, P < 0.05). Similar elevation in IL-10 plasma levels was detected in SPX DBA/2J mice as compared to NoOp DBA/2J mice, but not in C57BL/6J and BALB/cJ mice. In contrast, SPX AKR mice displayed lower IL-10 levels than NoOp mice. PMphis from SPX A/J mice produced elevated levels of IL-10 compared to PMphis from SHAM or NoOp A/J mice, mimicking the in vivo observations. CONCLUSION: Our data suggest that the spleen plays an important role in modulating the inflammatory process induced by LPS, extending beyond passive clearance of encapsulated organisms. In addition, the contribution of the spleen to the inflammatory process may be influenced by the genetic background.

Age and caloric restriction diets are confounding factors that modify the response to lipopolysaccharide by peritoneal macrophages in C57BL/6 mice.

Aging is the result of several detrimental changes that lead to a decrease in homeostasis, an increase in the incidence of degenerative diseases, and death. A caloric-restricted diet (CR), which consists of a significant reduction in calorie intake (40%) without malnutrition, has been shown to delay the onset of age-related diseases and pathologies and to extend life span. The aims of this study were to assess the effects of aging and CR on lipopolysaccharide (LPS)-dependant cytokine production by peritoneal macrophages (PMphis). Resident naïve PMphis were isolated from 2- to 24-month-old male C57BL/6 mice and were stimulated with Escherichia coli LPS (100 ng/mL) for 1 to 5 h in culture conditions. A linear decrease in the production of LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 10 was observed with age. LPS-induced IL-6 and IL-1beta levels were also reduced with age, but in a nonlinear fashion. Expression of CD14, the major receptor for LPS, on the PMphi surface was also observed to decline with age. Moreover, TNF-alpha production by PMphis was reduced in mice undergoing the two different CR diets of limited daily feeding and intermittent fasting, as compared with ad libitum-fed mice. The results of this study add the new variables age and diet to the paradigm proposing that the response to LPS is modulated by multiple components, including genetic background and sex.

Geldanamycin treatment ameliorates the response to LPS in murine macrophages by decreasing CD14 surface expression.

Geldanamycin (GA) is an antibiotic produced by Actinomyces, which specifically inhibits the function of the heat shock protein 90 family. Treatment of a murine macrophage cell line (J774) with GA resulted in a reduced response to Escherichia coli lipopolysaccharide (LPS) as visualized by a decrease of NF-kappaB translocation into the nucleus and secretion of tumor necrosis factor alpha (TNF-alpha). To elucidate the mechanism of this effect, the expression of CD14, the formal LPS receptor, was analyzed. Cells treated with GA showed a reduced level of surface CD14 detected by immunostaining, whereas the expression of other surface receptors, such as FC-gamma receptor and tumor necrosis factor receptors (TNF-R1 and TNF-R2), was unaffected. The reduced surface level of CD14 was not due to a reduction in its expression because CD14 steady state mRNA levels or the total cellular pool of CD14 was not altered by GA treatment. Surface CD14 was more rapidly internalized after GA treatment (2-3 h) than after incubation with cycloheximide. Immunostaining of permeabilized cells after GA treatment revealed a higher intracellular content of CD14 colocalizing with calnexin, an endoplasmic reticulum (ER) protein. These results suggest that the decrease in CD14 surface expression after GA treatment is due to rapid internalization without new replacement. These effects may be due to the inhibition of Hsp90 and Grp94 by GA in macrophages.

Gap junctions, homeostasis, and injury.

Gap junctions (Gj) play an important role in the communication between cells of many tissues. They are composed of channels that permit the passage of ions and low molecular weight metabolites between adjacent cells, without exposure to the extracellular environment. These pathways are formed by the interaction between two hemichannels on the surface of opposing cells. These hemichannels are formed by the association of six identical subunits, named connexins (Cx), which are integral membrane proteins. Cell coupling via Gj is dependent on the specific pattern of Cx gene expression. This pattern of gene expression is altered during several pathological conditions resulting in changes of cell coupling. The regulation of Cx gene expression is affected at different levels from transcription to post translational processes during injury. In addition, Gj cellular communication is regulated by gating mechanisms. The alteration of Gj communication during injury could be rationalized by two opposite theories. One hypothesis proposes that the alteration of Gj communication attenuates the spread of toxic metabolites from the injured area to healthy organ regions. The alternative proposition is that a reduction of cellular communication reduces the loss of important cellular metabolisms, such as ATP and glucose.