Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation.

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H- but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

Short Time to Market and Forward Planning Will Enable Cell Therapies to Deliver R&D Pipeline Value.

There is considerable industry excitement about the curative potential of cell and gene therapies, but significant challenges remain in designing cost-effective treatments that are accessible globally. We have taken a modeling-based approach to define the cost and value drivers for cell therapy assets during pharmaceutical drug development. We have created a model development program for a lentiviral modified ex vivo autologous T cell therapy for Oncology indications. Using internal and external benchmarks, we have estimated the total out-of-pocket cost of development for an Oncology cell therapy asset from target identification to filing of marketing application to be $500-600 million. Our model indicates that both clinical and Chemistry Manufacturing and Controls (CMC) cost of development for cell therapies are higher due to unique considerations of ex vivo autologous cell therapies. We have computed a threshold revenue-generating patient number for our model asset that enables selection of assets that can address high unmet medical need and generate pipeline value. Using statistical approaches, we identified that short time to market (<5 years) and reduced commercial cost of goods (<$65,000 per dose) will be essential in developing competitive assets and we propose solutions to reduce both. We emphasize that teams must proactively plan alternate development scenarios with clear articulation of path to value generation and greater patient access. We recommend using a modeling-based approach to enable data driven go/no-go decisions during multigenerational cell therapy development.

The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Oxygen and Conformation Dependent Protein Oxidation and Aggregation by Porphyrins in Hepatocytes and Light-Exposed Cells.

BACKGROUND & AIMS: Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent. METHODS: Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed. RESULTS: Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source. CONCLUSIONS: Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias.

Restoration of the basement membrane after wounding: a hallmark of young human skin altered with aging.

In skin, the basement membrane at the dermal-epidermal junction (DEJ-BM) is an important structure that tightly binds the epidermis to the dermis, and acts as a permeability barrier that controls exchange of macromolecules. Repair of the DEJ-BM during wound healing is important for restoration of skin functional properties after wounding. Here, we used a CO2 laser to perform partial thickness wounds in human volunteers, and directly compared wound repair in healthy young and aged individuals, focusing on the DEJ-BM. Our results show that the DEJ-BM is restored within four weeks after partial thickness wounds in young adults. We identified laminin-γ2 as preferred substrate for keratinocytes during reepithelialization of partial thickness human wounds. Laminin-γ2 is expressed continuously by migrating keratinocytes during reepithelialization, whereas collagen IV and collagen VII are deposited after wound closure. In contrast, our study shows that the DEJ-BM restoration following wounding is deficient in elderly individuals. Specifically, COL7A2 was barely increased during wound repair in aged skin and, as a result, the DEJ-BM in elderly skin was not restored and showed abnormal structure. Our data suggest that ameliorating the quality of the DEJ-BM restoration is a promising therapeutic approach to improve the quality of repaired skin in the elderly.

Mouse Models of Corneal Scarring.

Corneal scarring is an obligatory consequence of stroma corneal injury and is a major cause of decreased visual quality and vision loss worldwide. There are currently no satisfactory intervention therapies for corneal fibrosis. In this chapter, we describe well-established in vivo corneal wound models to allow researchers to investigate epithelial and stromal responses to corneal injury.

Cell-Populated Collagen Lattice Models.

Investigation of cell function is often hampered by the complexity of the tissue context. This problem is circumvented by isolating cells from tissues and analyzing their behavior in culture. Most cell types are cultured as monolayers on planar, rigid Petri dishes, an environment that does not reflect the spatial, three-dimensional cellular environment in vivo. Culture in three-dimensional collagen lattices has been devised to optimize in vitro culture conditions and to provide a more physiologic "in vivo-like" environment. Collagen lattices can easily be manipulated to suit diverse cell types and to provide variable mechanical forces. Cells can be imaged in such surroundings, and gene expression as well as protein production and activity can be monitored.

Mechanical Deformation of Cultured Cells with Hydrogels.

Polyacrylamide hydrogels can be used to culture cells in a range of stiffness that can closer mimic physiological environments. Changes in environmental stiffness have been documented in conditions such as fibrosis, cancer, and aging. In this chapter, we describe a method in which we pour gels directly into multiwell plates using a plastic support that covalently binds to the polymerizing hydrogel. The hydrogel is then crosslinked to calfskin collagen using a crosslinker. The result is a thick hydrogel, scalable to any size plate, which covers the entire surface of the well with no edge effects. The gels can be routinely assembled and are easily reproducible. These scaffolds are used as in vitro models to study fibroblast reaction to variation in environmental stiffness.

Type I Collagen Purification from Rat Tail Tendons.

Type I collagen, or collagen I, is the most abundant protein in the human body and provides strength and resiliency to tissues such as bone, tendons, ligaments, and skin. Collagen I forms macromolecular networks in which resident mesenchymal cells are embedded. Cell-extracellular matrix interactions are critical not only for maintenance of tissue properties but also for guiding and orienting the phenotype of resident cells. Cues from the extracellular matrix have been shown to be critical in pathophysiologies such as fibrosis, aging, and cancer. Hence, the details of these interactions are being scrutinized to better understand the mechanisms of such diseases and conditions. Many in vitro assays, such as cell-embedded collagen lattices, preparation of hydrogels, adhesion assays, etc., have been developed to study various aspects of cell-extracellular matrix interactions. All these in vitro models rely on utilizing high-quality purified collagen I. Here, we provide state-of-the-art collagen I extraction protocol and useful tips to produce high-quality purified collagen I solutions. We also provide a detailed protocol for pepsin digestion of collagen I, for a highly reliable collagen concentration assay, and guidelines for conducting quality controls to validate purified collagen solutions. Collagen I prepared with these procedures is highly suitable for many in vitro applications.

Method for Picrosirius Red-Polarization Detection of Collagen Fibers in Tissue Sections.

The extracellular matrix is critical in guiding cell behavior in normal and pathologic tissues, particularly in cancers and fibrosis. Highlighting the organization (or disorganization) of a collagen-containing matrix can be very useful for understanding or grading pathophysiological conditions. The picrosirius red stain (also called "Sirius red" stain) is one of the best understood histochemical techniques able to selectively highlight collagen networks. Relatively inexpensive, the technique relies on the birefringent properties of collagen molecules. While the picrosirius red stain alone does not selectively bind collagen network, it becomes more specific than the other common collagen stains when combined with polarized light detection. This is why the selective histochemical procedure for collagen detection should be called the picrosirius-polarization method. In this chapter, we will provide essential explanation and detailed protocols and tips to allow collagen researchers not only to better understand how this staining technique works but also to easily apply this technique to their collagen-related research.

Polycomb-Mediated Disruption of an Androgen Receptor Feedback Loop Drives Castration-Resistant Prostate Cancer.

The lethal phenotype of castration-resistant prostate cancer (CRPC) is generally caused by augmented signaling from the androgen receptor (AR). Here, we report that the AR-repressed gene CCN3/NOV inhibits AR signaling and acts in a negative feedback loop to block AR function. Mechanistically, a cytoplasmic form of CCN3 interacted with the AR N-terminal domain to sequester AR in the cytoplasm of prostate cancer cells, thereby reducing AR transcriptional activity and inhibiting cell growth. However, constitutive repression of CCN3 by the Polycomb group protein EZH2 disrupted this negative feedback loop in both CRPC and enzalutamide-resistant prostate cancer cells. Notably, restoring CCN3 was sufficient to effectively reduce CPRC cell proliferation in vitro and to abolish xenograft tumor growth in vivo Taken together, our findings establish CCN3 as a pivotal regulator of AR signaling and prostate cancer progression and suggest a functional intersection between Polycomb and AR signaling in CRPC. Cancer Res; 77(2); 412-22. ©2016 AACR.

Sebaceous Gland Atrophy in Psoriasis: An Explanation for Psoriatic Alopecia?

In a transcriptome study of lesional psoriatic skin (PP) versus normal skin, we found a coexpressed gene module (N5) enriched 11.5-fold for lipid biosynthetic genes. We also observed fewer visible hairs in PP skin, compared with uninvolved nonlesional psoriatic skin or normal skin (P < 0.0001). To ask whether these findings might be due to abnormalities of the pilosebaceous unit, we carried out three-dimensional morphometric analysis of paired PP and nonlesional psoriatic skin biopsies. Sebaceous glands were markedly atrophic in PP versus nonlesional psoriatic skin (91% average reduction in volume, P = 0.031). Module N5 genes were strongly downregulated in PP versus normal skin (fold change < 0.25, 44.4-fold) and strongly upregulated in sebaceous hyperplasia (fold change > 4, 54.1-fold). The intersection of PP-downregulated and sebaceous hyperplasia-upregulated gene lists generated a gene expression signature consisting solely of module N5 genes, whose expression in PP versus normal skin was inversely correlated with the signature of IL17-stimulated keratinocytes. Despite loss of visible hairs, morphometry identified elongated follicles in PP versus nonlesional psoriatic skin (average 1.7 vs. 1.2 µm, P = 0.020). These results document sebaceous gland atrophy in nonscalp psoriasis, identify a cytokine-regulated set of sebaceous gland signature genes, and suggest that loss of visible hair in PP skin may result from abnormal sebaceous gland function.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Cellular mechanisms of skin repair in humans and other mammals.

The increased incidence of non-healing skin wounds in developed societies has prompted tremendous research efforts on the complex process known as "wound healing". Unfortunately, the weak relevance of modern wound healing research to human health continues to be a matter of concern. This review summarizes the current knowledge of the cellular mechanisms that mediate wound closure in the skin of humans and laboratory animals. The author highlights the anatomical singularities of human skin vs. the skin of other mammals commonly used for wound healing research (i.e. as mice, rats, rabbits, and pigs), and discusses the roles of stem cells, myofibroblasts, and the matrix environment in the repair process. The majority of this review focuses on reepithelialization and wound closure. Other aspects of wound healing (e.g. inflammation, fibrous healing) are referred to when relevant to the main topic. This review aims at providing the reader with a clear understanding of the similarities and differences that have been reported over the past 100 years between the healing of human wounds and that of other mammals.

Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin.

Human skin heals more slowly in aged vs. young adults, but the mechanism for this delay is unclear. In humans, eccrine sweat glands (ESGs) and hair follicles underlying wounds generate cohesive keratinocyte outgrowths that expand to form the new epidermis. Here, we compared the re-epithelialization of partial-thickness wounds created on the forearm of healthy young (< 40 yo) and aged (> 70 yo) adults. Our results confirm that the outgrowth of cells from ESGs is a major feature of repair in young skin. Strikingly, in aged skin, although ESG density is unaltered, less than 50% of the ESGs generate epithelial outgrowths during repair (vs. 100% in young). Surprisingly, aging does not alter the wound-induced proliferation response in hair follicles or ESGs. Instead, there is an overall reduced cohesiveness of keratinocytes in aged skin. Reduced cell-cell cohesiveness was most obvious in ESG-derived outgrowths that, when present, were surrounded by unconnected cells in the scab overlaying aged wounds. Reduced cell-cell contact persisted during the repair process, with increased intercellular spacing and reduced number of desmosomes. Together, reduced outgrowths of ESG (i) reduce the initial number of cells participating in epidermal repair, (ii) delay wound closure, and (iii) lead to a thinner repaired epidermis in aged vs. young skin. Failure to form cohesive ESG outgrowths may reflect impaired interactions of keratinocytes with the damaged ECM in aged skin. Our findings provide a framework to better understand the mediators of delayed re-epithelialization in aging and further support the importance of ESGs for the repair of human wounds.

Matricellular protein CCN3 mitigates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease.

Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation.

The epidermal growth factor receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies.

Transgenic expression of human amphiregulin in mouse skin: inflammatory epidermal hyperplasia and enlarged sebaceous glands.

To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5' and 3' untranslated region sequences (AREG-UTR) led to a >10-fold increase in hAREG expression compared to an otherwise-identical construct containing only the coding region (AREG-CDR). AREG-UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG-CDR mice. Histologically, AREG-UTR mice presented with marked epidermal hyperplasia of tail skin (2.1-fold increase in epidermal thickness with a 9.5-fold increase in Ki-67(+) cells) accompanied by significantly increased CD4+ T-cell infiltration. Dorsal skin of AREG-UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG-UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki-67(+) cells. To determine the response of AREG-UTR animals to a pro-inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG-UTR and wild type mice (1.7- and 2.2-fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen.

Another dimension to the importance of the extracellular matrix in fibrosis.

The importance of the extracellular matrix (ECM) in fibrosis has been recognized for a long time, not only because ECM's increased stiffness hampers tissue function, but also because the ECM provides the mechanical tension that maintains resident cells' synthetic phenotype. A study by Parker and colleagues (Journal of Clinical Investigation 124, 1622-1635, 2014) compared the transcriptome of fibroblasts cultured on decellularized ECM from healthy vs idiopathic pulmonary fibrosis (IPF) human lungs, and revealed that the IPF matrix exerts a positive feedback loop that increases the translation of ECM genes that are enriched in the IPF ECM proteome. This study suggests that the ECM composition, in addition to ECM stiffness or the phenotype of its resident cells, might be an important factor in maintaining a fibrotic state. Targeting this feedback loop might be an efficient therapeutic strategy for IPF.