Unblinding in Randomized Controlled Trials: A Research Ethics Case.

A pregnant woman was enrolled in a double-blind randomized controlled trial (RCT) in which participants were randomized to a placebo or a drug being tested to prevent a hypertensive complication. After completing the trial, the research participant insisted on being told which drug she received to prepare for a future pregnancy. This case highlights an element of RCT procedure that has received minimal attention-whether to unblind study participants at the end of their participation. Given that unblinding is not standard practice for nonserious adverse events, what actions are justifiable for the investigators to take? To synthesize the information about this case, we used the CASES model, created by the National Center for Ethics in Health Care to analyze ethics cases. Ethical principles that guide research emphasize communication with participants and the importance of reducing harm within the constraints of the scientific goals. Participants may value knowing which drug they received for future health care decision-making. We review information about the benefits and harms of unblinding.

Wound Healing Driver Gene and Therapeutic Development: Political and Scientific Hurdles.

Significance: Since the last Food and Drug Administration (FDA) approval of a wound healing therapeutic in 1997, no new therapeutic candidate (excluding physical therapies, devices, dressings, and antimicrobial agents) has advanced to clinical applications. During this period, the FDA drug approvals for tumors, which have been referred to as "wounds that do not heal," have reached a total of 284 (by end of 2018). Both political and scientific factors may explain this large discrepancy in drug approvals for the two seemingly related and equally complex pathophysiological conditions. Recent Advances: Using the current research funding ratio of 1:150 for wound healing to cancer and the 5% FDA drug approval rate for oncology, we reach a crude estimate of a 0.03% success rate for wound healing therapeutics. Unless a drastic improvement of the current situation, we express a pessimistic outlook toward new and effective wound healing drugs. Critical Issues: We argue that successful development of wound healing therapeutics will rely on identification of wound healing driver genes (WDGs), and the focus should be on WDGs for the wound closure phase of wound healing. Therefore, WDGs must be both necessary and sufficient for wound closure; the absence of a WDG disrupts wound closure, while its supplementation alone is sufficient to restore full wound closure. Successful translation of a WDG into therapeutics requires availability of well-defined animal models with a high degree of relevance to humans. This review discusses the main hurdles faced by the wound healing research community behind the development of so-called "rescuing drugs" for wound healing. Future Directions: Given the lack of new wound healing drugs for the past 23 years, there is a need for a wide range of fresh, innovative, and thorough debates on wound healing drug development, including an organized movement to raise public support for wound healing research.

Extracellular and Non-Chaperone Function of Heat Shock Protein-90α Is Required for Skin Wound Healing.

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs.

Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5-treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing.

Keratinocyte-Secreted Heat Shock Protein-90alpha: Leading Wound Reepithelialization and Closure.

Significance: Delayed and nonhealing wounds pose a health, economic, and social problem worldwide. For decades, the conventional wisdom pointed to growth factors as the driving force of wound healing and granted them a center stage for therapeutic development. To date, few have obtained US FDA approvals or shown clinical effectiveness and safety. Critical Issue: Wound closure is the initial and most critical step during wound healing. Closing chronic wounds to shut down continued infection is the primary and likely the only achievable goal at the clinic in the foreseeable future. The critical question here is to identify the factor(s) in wounded tissues that drives the initial wound closure. Recent Advances: We made an unexpected discovery of the secreted form of heat shock protein-90alpha (Hsp90α) for promoting skin cell motility, reepithelialization, and wound closure. Secreted Hsp90α possesses unique properties to remain functional under the hostile wound environment that compromises conventional growth factors' effectiveness. Through the common lipoprotein receptor-related protein-1 cell surface receptor and activation of the Akt signaling pathway, topical application of human recombinant Hsp90α protein greatly accelerates excision, burn, and diabetic skin wound closure in rodent and porcine models. Future Directions: In almost all cells, the 2-3% of their total proteins (∼7,000 per cell) are Hsp90 (α and ß), a long unraveled puzzle. Our new finding of Hsp90 secretion in wounded tissues suggests that the stockpile of Hsp90α by all cells is to rapidly supply the need for extracellular Hsp90α to repair damaged tissues. We propose that keratinocytes at the wound edge secrete Hsp90α that leads the reepithelialization process to close the wound.

Breast Cancer MDA-MB-231 Cells Use Secreted Heat Shock Protein-90alpha (Hsp90α) to Survive a Hostile Hypoxic Environment.

Rapidly growing tumours in vivo often outgrow their surrounding available blood supply, subjecting themselves to a severely hypoxic microenvironment. Understanding how tumour cells adapt themselves to survive hypoxia may help to develop new treatments of the tumours. Given the limited blood perfusion to the enlarging tumour, whatever factor(s) that allows the tumour cells to survive likely comes from the tumour cells themselves or its associated stromal cells. In this report, we show that HIF-1α-overexpressing breast cancer cells, MDA-MB-231, secrete heat shock protein-90alpha (Hsp90α) and use it to survive under hypoxia. Depletion of Hsp90α secretion from the tumour cells was permissive to cytotoxicity by hypoxia, whereas supplementation of Hsp90α-knockout tumour cells with recombinant Hsp90α, but not Hsp90ß, protein prevented hypoxia-induced cell death via an autocrine mechanism through the LDL receptor-related protein-1 (LRP1) receptor. Finally, direct inhibition of the secreted Hsp90α with monoclonal antibody, 1G6-D7, enhanced tumour cell death under hypoxia. Therefore, secreted Hsp90α is a novel survival factor for certain tumours under hypoxia.

Hsp90α and Hsp90ß together operate a hypoxia and nutrient paucity stress-response mechanism during wound healing.

When tissues are injured and blood vessels clot, the local environment becomes ischemic, meaning that there is a lack of adequate supply of oxygen and glucose delivered to the surrounding cells. The heat shock protein-90 (Hsp90) family proteins protect tissues from various environmental insults and participate in the repair of damaged tissue. Here, we report discovery of a new ischemia-responsive mechanism in which the two Hsp90 isoforms Hsp90α and Hsp90ß (also known as HSP90AA1 and HSP90AB1, respectively) work together to promote cell motility in wounded skin and accelerate wound closure. We demonstrate that Hsp90α and Hsp90ß have distinct and non-exchangeable functions during wound healing. Under hypoxia and when there is a lack of serum factors, Hsp90ß binds to the cytoplasmic tail of the LDL receptor-related protein-1 (LRP-1) and stabilizes the receptor at the cell surface. Hsp90α, however, is secreted by the cell into extracellular space where it binds and signals through the LRP-1 receptor to promote cell motility, leading to wound closure. In addition to skin injury, we suggest that this repair mechanism applies broadly to other non-cutaneous injured tissues.

Identification of the critical therapeutic entity in secreted Hsp90α that promotes wound healing in newly re-standardized healthy and diabetic pig models.

Chronic and non-healing skin wounds represent a significant clinical, economic and social problem worldwide. Currently, there are few effective treatments. Lack of well-defined animal models to investigate wound healing mechanisms and furthermore to identify new and more effective therapeutic agents still remains a major challenge. Pig skin wound healing is close to humans. However, standardized pig wound healing models with demonstrated validity for testing new wound healing candidates are unavailable. Here we report a systematic evaluation and establishment of both acute and diabetic wound healing models in pigs, including wound-creating pattern for drug treatment versus control, measurements of diabetic parameters and the time for detecting delayed wound healing. We find that treatment and control wounds should be on the opposite and corresponding sides of a pig. We demonstrate a strong correlation between duration of diabetic conditions and the length of delay in wound closure. Using these new models, we narrow down the minimum therapeutic entity of secreted Hsp90α to a 27-amino acid peptide, called fragment-8 (F-8). In addition, results of histochemistry and immunohistochemistry analyses reveal more organized epidermis and dermis in Hsp90α-healed wounds than the control. Finally, Hsp90α uses a similar signaling mechanism to promote migration of isolated pig and human keratinocytes and dermal fibroblasts. This is the first report that shows standardized pig models for acute and diabetic wound healing studies and proves its usefulness with both an approved drug and a new therapeutic agent.

Extracellular heat shock protein 90 signals through subdomain II and the NPVY motif of LRP-1 receptor to Akt1 and Akt2: a circuit essential for promoting skin cell migration in vitro and wound healing in vivo.

Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.

Extracellular Hsp90 (eHsp90) as the actual target in clinical trials: intentionally or unintentionally.

Despite extensive investigative studies and clinical trials over the past two decades, we still do not understand why cancer cells are more sensitive to the cellular toxicity of Hsp90 inhibitors than normal cells. We still do not understand why only some cancer cells are sensitive to the Hsp90 inhibitors. Based on studies of the past few years, we argue that the selected sensitivity of cancer cells to Hsp90 inhibitors, such as 17-N-allylamino-17-demethoxygeldanamycin, is due to inhibition of the extracellular Hsp90 (eHsp90) rather than intracellular Hsp90 by these inhibitors. Because not all tumor cells utilize eHsp90 for motility, invasion and metastasis, only the group of "eHsp90-dependent" cancer cells is sensitive to Hsp90 inhibitors. If these notions prove to be true, pharmaceutical agents that selectively target eHsp90 should be more effective on tumor cells and less toxic on normal cells than current inhibitors that nondiscriminatively target both extracellular and intracellular Hsp90.

TPL2/COT/MAP3K8 (TPL2) activation promotes androgen depletion-independent (ADI) prostate cancer growth.

BACKGROUND: Despite its initial positive response to hormone ablation therapy, prostate cancers invariably recur in more aggressive, treatment resistant forms. The lack of our understanding of underlying genetic alterations for the transition from androgen-dependent (AD) to ADI prostate cancer growth hampers our ability to develop target-driven therapeutic strategies for the efficient treatment of ADI prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: By screening a library of activated human kinases, we have identified TPL2, encoding a serine/threonine kinase, as driving ADI prostate cancer growth. TPL2 activation by over-expressing either wild-type or a constitutively activated form of TPL2 induced ADI growth, whereas the suppression of TPL2 expression and its kinase activity in ADI prostate cancer cells inhibited cell proliferation under androgen-depleted conditions. Most importantly, TPL2 is upregulated in ADI prostate cancers of both the Pten deletion mouse model and the clinical prostate cancer specimens. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that TPL2 kinase plays a critical role in the promotion of ADI prostate cancer progression. Furthermore, the suppression of TPL2 diminishes ADI prostate cancer growth and a high frequency of TPL2 overexpression in human ADI prostate cancer samples validates TPL2 as a target for the treatment of this deadly disease.