AGSE: A Novel Grape Seed Extract Enriched for PP2A Activating Flavonoids That Combats Oxidative Stress and Promotes Skin Health.

Environmental stimuli attack the skin daily resulting in the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein Phosphatase 2A (PP2A), a phosphatase which has been previously linked to Alzheimer's Disease and aging. Oxidative stress decreases PP2A methylation in normal human dermal fibroblasts (NHDFs). Thus, we hypothesize agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a library of 32 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin, to be the most potent PP2A demethylating extract. Via several fractionation and extraction steps we developed a novel grape seed extract called Activated Grape Seed Extract (AGSE), which is enriched for PP2A activating flavonoids that increase potency in preventing PP2A demethylation when compared to commercial GSE. We then determined that 1% AGSE and 1% commercial GSE exhibit distinct gene expression profiles when topically applied to a 3D human skin model. To begin to characterize AGSE's activity, we investigated its antioxidant potential and demonstrate it reduces ROS levels in NHDFs and cell-free assays equal to or better than Vitamin C and E. Moreover, AGSE shows anti-inflammatory properties, dose-dependently inhibiting UVA, UVB and chemical-induced inflammation. These results demonstrate AGSE is a novel, multi-functional extract that modulates methylation levels of PP2A and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin.

HYVIA™: A novel, topical chia seed extract that improves skin hydration.

BACKGROUND: Chia seeds have gained importance as it is the highest known plant source of omega-3 (ω3) polyunsaturated fatty acids. Specifically, chia seeds possess ω3 α-linolenic acid (ALA) and ω6 linoleic acid (LA), together known as Vitamin F, which play an important role in maintaining skin function. Protein phosphatase 2A (PP2A) is a master regulatory protein that plays a critical role in skin barrier function and its activity is modulated by natural lipids. AIMS: Obtain a chia seed extract (HYVIA™) with significant higher levels of Vitamin F, determine in vitro PP2A activity and skin hydration markers compared to other commercial chia seed extracts (CCSEs), and evaluate the potential skin hydration benefits clinically in human subjects. METHODS: A PP2A demethylation assay was utilized to assess PP2A activity. In vitro studies utilizing normal human epidermal keratinocytes (NHEKs) were treated with HYVIA™ and gene expression of hydration markers (AQP3, HAS2) were measured by quantitative PCR (qPCR). A 16-subject clinical trial was performed with 0.1% HYVIA™ formulated in a cream and applied topically to assess its skin moisturizing potential. RESULTS: We demonstrate here that HYVIA™, ALA, and LA inhibit PP2A demethylation, boosting PP2A activity, while most other CCSEs do not. Unlike other CCSEs, HYVIA™ increases keratinocyte hydration factors aquaporin-3 and hyaluronic acid synthase-2 in vitro. Clinical assessment of 0.1% HYVIA™ cream shows that HYVIA™ improves skin hydration. CONCLUSIONS: HYVIA™ is a novel chia seed extract, enriched for Vitamin F, that modulates PP2A activity and clinically improves skin hydration and barrier function.

SIG-1273 protects skin against urban air pollution and when formulated in AgeIQ™ Night Cream anti-aging benefits clinically demonstrated.

BACKGROUND: SIG-1273 is a novel cosmetic active that provides a broad spectrum of benefits to the skin. Considering the chronic skin exposure to pollution in urban areas, we sought to determine if SIG-1273 could provide additional protection against skin aging by inhibiting pollutant-induced cytotoxicity and inflammation. OBJECTIVE: Determine if SIG-1273 possesses antipollution properties in vitro and evaluate the potential anti-aging benefits of Age IQ™ Night Cream clinically in human subjects. METHODS: In vitro studies utilizing normal human epidermal keratinocytes (NHEKs), were co-treated with urban dust (SRM 1649b) and SIG-1273 (toxicity protection measured by MTS assay). A water-soluble fraction of urban dust (UD-WS) induces pro-inflammatory cytokine release (IL-8) from NHEKs (measured via ELISA). An 8-week, 37-subject clinical trial was performed with 0.05% SIG-1273 formulated in Age IQ™ Night Cream and applied topically to assess its potential to reduce the appearance of aging. RESULTS: In vitro studies using NHEKs demonstrate SIG-1273 protects against urban dust-induced cell toxicity, reducing cell death by 66% and concentration dependently inhibits UD-WS-induced IL-8 production (IC50  = 20 nmol/L), outperforming niacinamide, ascorbic acid, and α-tocopherol, commonly used actives in antipollution skin-care products. Clinical assessment of Age IQ™ Night Cream shows it is effective in improving the appearance of facial skin aging including fine lines and wrinkles, skin texture, skin clarity/brightness, and firmness/elasticity. CONCLUSIONS: SIG-1273, is demonstrated here for the first time to possess antipollution properties. Included as a key active ingredient in Age IQ™ Night Cream, this novel topical formulation provides benefits to individuals with aging skin.

SIG1459: A novel phytyl-cysteine derived TLR2 modulator with in vitro and clinical anti-acne activity.

Cutibacterium (formerly Propionibacterium acnes) is a major contributor to the pathogenesis of acne. C. acnes initiates an innate immune response in keratinocytes via recognition and activation of toll-like receptor-2 (TLR2), a key step in comedogenesis. Tetramethyl-hexadecenyl-cysteine-formylprolinate (SIG1459), a novel anti-acne isoprenylcysteine (IPC) small molecule, is shown in this study to have direct antibacterial activity and inhibit TLR2 inflammatory signalling. In vitro antibacterial activity of SIG1459 against C. acnes was established demonstrating minimal inhibitory concentration (MIC = 8.5 µmol\L), minimal bactericidal concentration (MBC = 16.1 µmol\L) and minimal biofilm eradication concentration (MBEC = 12.5 µmol\L). To assess SIG1459's anti-inflammatory activity, human keratinocytes were exposed to C. acnes and different TLR2 ligands (peptidoglycan, FSL-1, Pam3CSK4) that induce pro-inflammatory cytokine IL-8 and IL-1α production. Results demonstrate SIG1459 inhibits TLR2-induced IL-8 release from TLR2/TLR2 (IC50  = 0.086 µmol\L), TLR2/6 (IC50  = 0.209 µmol\L) and IL-1α from TLR2/TLR2 (IC50  = 0.050 µmol\L). To assess the safety and in vivo anti-acne activity of SIG1459, a vehicle controlled clinical study was conducted applying 1% SIG1459 topically (n = 35 subjects) in a head-to-head comparison against 3% BPO (n = 15 subjects). Utilizing the Investigator Global Assessment scale for acne as primary endpoint, results demonstrate 1% SIG1459 significantly outperformed 3% BPO over 8 weeks, resulting in 79% improvement as compared to 56% for BPO. Additionally, 1% SIG1459 was well tolerated. Thus, SIG1459 and phytyl IPC compounds represent a novel anti-acne technology that provides a safe dual modulating benefit by killing C. acnes and reducing the inflammation it triggers via TLR2 signalling.

N-Acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191): an anti-inflammatory molecule that increases the expression of the aquaglyceroporin, aquaporin-3, in human keratinocytes.

Isoprenylcysteine (IPC) small molecules were discovered as signal transduction modulating compounds ~25 years ago. More recently, IPC molecules have demonstrated antioxidant and anti-inflammatory properties in a variety of dermal cells as well as antimicrobial activity, representing a novel class of compounds to ameliorate skin conditions and disease. Here, we demonstrate a new IPC compound, N-acetylglutaminoyl-S-farnesyl-L-cysteine (SIG-1191), which inhibits UVB-induced inflammation blocking pro-inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) production. To investigate further the previously reported hydrating potential of IPC compounds, SIG-1191 was tested for its ability to modulate aquaporin expression. Specifically, aquaporin 3 (AQP3) the most abundant aquaporin found in skin has been reported to play a key role in skin hydration, elasticity and barrier repair. Results show here for the first time that SIG-1191 increases AQP3 expression in both cultured normal human epidermal keratinocytes as well as when applied topically in a three-dimensional (3D) reconstructed human skin equivalent. Additionally, SIG-1191 dose dependently increased AQP3 protein levels, as determined by specific antibody staining, in the epidermis of the 3D skin equivalents. To begin to elucidate which signaling pathways SIG-1191 may be modulating to increase AQP3 levels, we used several pharmacological pathway inhibitors and determined that AQP3 expression is mediated by the Mitogen-activated protein kinase/Extracellular signal-regulated kinase kinase (MEK) pathway. Altogether, these data suggest SIG-1191 represents a new IPC derivative with anti-inflammatory activity that may also promote increased skin hydration based on its ability to increase AQP3 levels.

Fat-water MRI of a diet-induced obesity mouse model at 15.2T.

Quantitative fat-water MRI (FWMRI) methods provide valuable information about the distribution, volume, and composition of adipose tissue (AT). Ultra high field FWMRI of animal models may have the potential to provide insights into the progression of obesity and its comorbidities. Here, we present quantitative FWMRI with all known confounder corrections on a 15.2T preclinical scanner for noninvasive in vivo monitoring of an established diet-induced obesity mouse model. Male C57BL/6J mice were placed on a low-fat (LFD) or a high-fat diet (HFD). Three-dimensional (3-D) multiple gradient echo MRI at 15.2T was performed at baseline, 4, 8, 12, and 16 weeks after diet onset. A 3-D fat-water separation algorithm and additional processing were used to generate proton-density fat fraction (PDFF), local magnetic field offset, and [Formula: see text] maps. We examined these parameters in perirenal AT ROIs from LFD and HFD mice. The data suggest that PDFF, local field offset, and [Formula: see text] have different time course behaviors between LFD and HFD mice over 16 weeks. This work suggests FWMRI at 15.2T may be a useful tool for longitudinal studies of adiposity due to the advantages of ultra high field although further investigation is needed to understand the observed time course behavior.

In vitro and clinical evaluation of SIG1273: a cosmetic functional ingredient with a broad spectrum of anti-aging and antioxidant activities.

BACKGROUND: Isoprenylcysteine (IPC) small molecules were identified as a new class of anti-inflammatory compounds over 20 years ago. Since then, they have been developed as novel cosmetic functional ingredients (CFI) and topical drug candidates. SIG1273 is a second generation CFI that has previously been shown to provide a broad spectrum of benefits for the skin through its anti-inflammatory and antimicrobial properties. OBJECTIVE: To determine whether SIG1273 possesses anti-aging properties in vitro and evaluate the tolerability and activity of SIG1273 when applied topically to human subjects. METHODS: To model photoaging in vitro, human dermal fibroblasts (HDFs) were exposed in culture to UVA to induce collagenase (MMP-1) production. An in vitro wound-healing model was based on the activation of HDF migration into cell-free tissue culture surface. Hydrogen peroxide-induced oxidative stress was performed using HDFs to measure intracellular ROS activity. Radical scavenging capacity was determined using a colorimetric antioxidant assay kit (ABTS method). Lastly, a 4-week, 29-subject study was performed in which SIG1273 was applied topically as a cream to assess its tolerance and activity in reducing the appearance of aging. RESULTS: In vitro studies demonstrate SIG1273 inhibits UVA-induced MMP-1 production, hydrogen peroxide-induced oxidative stress and promotes wound healing. Moreover, SIG1273 was shown to be a radical scavenging antioxidant. Clinical assessment of SIG1273 cream (0.25%) showed it was well tolerated with significant improvement in the appearance of fine lines, coarse wrinkles, radiance/luminosity, pore size, texture/smoothness, hydration and increased firmness. CONCLUSIONS: SIG1273 represents a novel CFI with antioxidant, anti-aging, and anti-inflammatory properties that when applied topically is well tolerated and provides benefits to individuals with aging skin.

Activation of NF-κB drives the enhanced survival of adipose tissue macrophages in an obesogenic environment.

OBJECTIVE: Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and insulin resistance. Recruitment of monocytes to obese AT has been the most studied mechanism explaining this accumulation. However, recent evidence suggests that recruitment-independent mechanisms may also regulate pro-inflammatory AT macrophage (ATM) numbers. The role of increased ATM survival during obesity has yet to be explored. RESULTS: We demonstrate that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria is elevated in ATMs from obese mice. This increased pro-survival signaling was associated with elevated activation of the transcription factor, NF-κB, and increased expression of its pro-survival target genes. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional. CONCLUSIONS: Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism.

Obesity alters adipose tissue macrophage iron content and tissue iron distribution.

Adipose tissue (AT) expansion is accompanied by the infiltration and accumulation of AT macrophages (ATMs), as well as a shift in ATM polarization. Several studies have implicated recruited M1 ATMs in the metabolic consequences of obesity; however, little is known regarding the role of alternatively activated resident M2 ATMs in AT homeostasis or how their function is altered in obesity. Herein, we report the discovery of a population of alternatively activated ATMs with elevated cellular iron content and an iron-recycling gene expression profile. These iron-rich ATMs are referred to as MFe(hi), and the remaining ATMs are referred to as MFe(lo). In lean mice, ~25% of the ATMs are MFe(hi); this percentage decreases in obesity owing to the recruitment of MFe(lo) macrophages. Similar to MFe(lo) cells, MFe(hi) ATMs undergo an inflammatory shift in obesity. In vivo, obesity reduces the iron content of MFe(hi) ATMs and the gene expression of iron importers as well as the iron exporter, ferroportin, suggesting an impaired ability to handle iron. In vitro, exposure of primary peritoneal macrophages to saturated fatty acids also alters iron metabolism gene expression. Finally, the impaired MFe(hi) iron handling coincides with adipocyte iron overload in obese mice. In conclusion, in obesity, iron distribution is altered both at the cellular and tissue levels, with AT playing a predominant role in this change. An increased availability of fatty acids during obesity may contribute to the observed changes in MFe(hi) ATM phenotype and their reduced capacity to handle iron.

Loss of CCR5 results in glucose intolerance in diet-induced obese mice.

Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C chemokine receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of proinflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5-deficient (CCR5(-/-)) mice and C57BL/6 (WT) controls were fed 10% low-fat (LF) or 60% high-fat (HF) diets for 16 wk. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5(-/-) mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1-associated gene CD11c, whereas M2 associated genes were not different between genotypes. In addition, HF feeding caused a twofold increase in CD4(+) T cells in the AT of CCR5(-/-) compared with WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However, in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRß in CCR5(-/-) compared with WT mice. These findings suggest that whereas CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

Fish oil and indomethacin in combination potently reduce dyslipidemia and hepatic steatosis in LDLR(-/-) mice.

Fish oil (FO) is a potent anti-inflammatory and lipid-lowering agent. Because inflammation can modulate lipid metabolism and vice versa, we hypothesized that combining FO with cyclooxygenase inhibitors (COXIBs), well-known anti-inflammatory drugs, can enhance the anti-inflammatory and lipid-lowering effect of FO. LDLR(-/-) mice were fed a high-fat diet supplemented with 6% olive oil or FO for 12 wk in the presence or absence of indomethacin (Indo, 6 mg/l drinking water). FO reduced plasma total cholesterol by 30% but, in combination with Indo, exerted a greater decrease (44%). The reduction of liver cholesterol ester (CE) and triglycerides (TG) by FO (63% and 41%, respectively) was enhanced by Indo (80% in CE and 64% in TG). FO + Indo greatly increased the expression of genes modulating lipid metabolism and reduced the expression of inflammatory genes compared with control. The mRNA and/or protein expression of pregnane X receptor (PXR) and cytochrome P450 isoforms that alter inflammation and/or lipid metabolism are increased to a greater extent in mice that received FO + Indo. Moroever, the nuclear level of PXR is significantly increased in FO + Indo group. Combining FO with COXIBs may exert their beneficial effects on inflammation and lipid metabolism via PXR and cytochrome P450.

Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion.

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR(-/-)) mice were transplanted with bone marrow from CCL3(-/-) or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3(-/-) marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3(-/-) recipients after 6 weeks and in male CCL3(-/-) recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3(-/-) recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR(-/-) recipients of monocyte chemoattractant protein(-/-) (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR(-/-) recipients of CCL3(-/-) marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR(-/-) controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3(-/-) recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution.

Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity.

OBJECTIVE: Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue-specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance. RESEARCH DESIGN AND METHODS: Mice with global or hematopoietic CCR2 deficiency (CCR2(-/-) and BM-CCR2(-/-), respectively) and wild-type controls (CCR2(+/+) and BM-CCR2(+/+), respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed. RESULTS: Flow cytometry analysis showed that two different populations of F4/80(+) myeloid cells (CD11b(lo)F4/80(lo) and CD11b(hi)F4/80(hi)) accumulated in the adipose tissue of CCR2(-/-) and BM-CCR2(-/-) mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b(hi)F4/80(hi) population was detected in the CCR2(+/+) and BM-CCR2(+/+) controls. After 20 weeks of HFD feeding, the CD11b(lo)F4/80(lo) cells were no longer present in the adipose tissue of CCR2(-/-) mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11b(lo)F4/80(lo) cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b(lo)F4/80(lo) cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue. CONCLUSIONS: The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.

Absence of macrophage inflammatory protein-1{alpha} does not impact macrophage accumulation in adipose tissue of diet-induced obese mice.

Macrophages and T-lymphocytes are known to accumulate in the white adipose tissue (WAT) of obese mice and humans, but the factors that cause this infiltration are not yet determined. Chemokines, which attract leukocytes to inflammatory sites, are candidates for this process. Macrophage inflammatory protein-1alpha (MIP-1alpha) expression is significantly elevated in WAT of obese mice and humans and positively correlates with fasting plasma insulin, but its potential role in leukocyte recruitment to WAT is unknown. MIP-1alpha-deficient, heterozygous, and wild-type mice were fed a Western diet (WD) for 16 wk. Plasma lipids, adipose tissue mass, energy expenditure, food intake, liver triglyceride content, and inflammatory cytokine expression were not different among genotypes. Gene expression of macrophage markers F4/80 and CD68, as well as T-lymphocyte marker CD3epsilon was increased in perigonadal WAT of obese WD-fed mice but was not influenced by MIP-1alpha expression level. Immunohistochemical analysis of WAT also showed no effect of MIP-1alpha on macrophage content. Two related chemokines, MIP-1beta and RANTES, had reduced expression in obese male MIP-1alpha-deficient mice compared with wild-type controls (P < or = 0.05). In mice fed the WD for 6 wk, WAT macrophage content was unchanged; however, CD8+ T-lymphocytes accumulated to a lesser extent in the MIP-1alpha-null mice. Therefore, expression of MIP-1alpha, as well as that of MIP-1beta and RANTES, increases as a consequence of weight gain, but these chemokines may not be required for the recruitment of monocytes to WAT during diet-induced obesity in mice and may impact T-lymphocyte recruitment only at early time points after WD feeding.