Amar Desai

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal.

Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science, this issue 10.1126/science.aaa2340; see also p. 1208 A compound that inhibits prostaglandin degradation enhances tissue regeneration in multiple organs in mice. [Also see Perspective by FitzGerald] INTRODUCTION Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. To date, therapeutic interventions have largely focused on targeting two PGE2 biosynthetic enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), with the aim of reducing PGE2 production. In this study, we take the converse approach: We examine the role of a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as a negative regulator of tissue repair, and we explore whether inhibition of this enzyme can potentiate tissue regeneration in mouse models. RATIONALE We used 15-PGDH knockout mice to elucidate the role of 15-PGDH in regulating tissue levels of PGE2 and tissue repair capacity in multiple organs. We then developed SW033291, a potent small-molecule inhibitor of 15-PGDH with activity in vivo. We used SW033291 to investigate the therapeutic potential of 15-PGDH inhibitors in tissue regeneration and to identify a 15-PGDH–regulated hematopoietic pathway within the bone marrow niche. RESULTS We found that in comparison with wild-type mice, 15-PGDH–deficient mice display a twofold increase in PGE2 levels across multiple tissues—including bone marrow, colon, and liver—and that they show increased fitness of these tissues in response to damage. The mutant mice also show enhanced hematopoietic capacity, with increased neutrophils, increased bone marrow SKL (Sca-1+ C-kit+ Lin−) cells (enriched for stem cells), and greater capacity to generate erythroid and myeloid colonies in cell culture. The 15-PGDH–deficient mice respond to colon injury from dextran sulfate sodium (DSS) with a twofold increase in cell proliferation in colon crypts, which confers resistance to DSS-induced colitis. The mutant mice also respond to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation, which leads to accelerated and more extensive liver regeneration. SW033291, a potent small-molecule inhibitor of 15-PGDH (inhibitor dissociation constant Ki ~0.1 nM), recapitulates in mice the phenotypes of 15-PGDH gene knockout, inducing increased hematopoiesis, resistance to DSS colitis, and more rapid liver regeneration after partial hepatectomy. Moreover, SW033291-treated mice show a 6-day-faster reconstitution of hematopoiesis after bone marrow transplantation, with accelerated recovery of neutrophils, platelets, and erythrocytes, and greater recovery of bone marrow SKL cells. This effect is mediated by bone marrow CD45– cells, which respond to increased PGE2 with a fourfold increase in production of CXCL12 and SCF, two cytokines that play key roles in hematopoietic stem cell homing and maintenance. CONCLUSIONS Studying mouse models, we have shown that 15-PGDH negatively regulates tissue regeneration and repair in the bone marrow, colon, and liver. Of most direct utility, our observations identify 15-PGDH as a therapeutic target and provide a chemical formulation, SW033291, that is an active 15-PGDH inhibitor in vivo and that potentiates repair in multiple tissues. SW033291 or related compounds may merit clinical investigation as a strategy to accelerate recovery after bone marrow transplantation and other tissue injuries. Inhibiting 15-PGDH accelerates tissue repair. (A) The enzyme 15-PGDH degrades and negatively regulates PGE2. (B) SW033291 inhibits 15-PGDH, increases tissue levels of PGE2, and induces CXCL12 and SCF expression from CD45– bone marrow cells. This in turn accelerates homing of transplanted hematopoietic stem cells (HSC), generation of mature blood elements, and post-transplant recovery of normal blood counts. Inhibiting 15-PGDH similarly stimulates cell proliferation after injury to colon or liver, accelerating repair of these tissues. Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

BACKGROUND Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. MATERIALS AND METHODS A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes. RESULTS A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. CONCLUSIONS CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.

Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long‐term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5′ HR end resection. We use cultured Exo1mut fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1mut mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1mut mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1mut HSCs were pushed into cell cycle in vivo with 5‐fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1‐mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. Stem Cells 2014;32:582–593

Advances in therapeutic targeting of the DNA damage response in cancer

The DNA damage response (DDR) is a series of pathways and processes required to repair lesions to DNA. These pathways range from repairing strand breaks to the double helix, damaged bases formed after oxidation or deamination, inaccurate DNA replication resulting in mispaired base alignment, intrastrand crosslinks that trigger cell death, and a plethora of other genomic insults. The DDR is believed to be a critical component of radio and chemoresistance in many cancers as well, with the tumors ability to repair therapy induced damage being an important tool used to survive traditional chemotherapeutic agents. Here we summarize advances made in specifically targeting DDR proteins in cancer therapy and project on the potential breakthroughs and pitfalls to arise as the field progresses.

Lentiviruses: Vectors for Cancer Gene Therapy

Lentivirus are the most efficient viral gene transfer vectors. Partitioned engineered backbones containing the essential proteins needed for reverse transcription and integration and separate elements for the transgene payload provide a 3 or 4 safety designed components that when transduced into transient producer cells yield high titre vectors. Applications of these vector systems have been designed for application for suicide gene therapy using thymidine kinase, immunotherapy and vaccine development, gene replacement and gene silencing including RNAi, anti-angiogenesis, an myelosuppression protection studies are discussed. Most of these efforts have moved from basic concept through preclinical testing and many are in early phase clinical trials. Lentiviral backbones remain a very promising approach to safe and stable gene transfer.

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation

One of the major health concerns on long‐duration space missions will be radiation exposure to the astronauts. Outside the earths magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation‐sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56Fe radiation on HSC function in Mlh1+/+ and Mlh1‐/‐ marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018;7:513–520

Concise Reviews: Cancer Stem Cell Targeted Therapies: Toward Clinical Success

Cancer stem cells (CSCs) are a subpopulation of cells within tumors that possess the stem cell characteristics of self‐renewal, quiescence, differentiation, and the ability to recapitulate the parental tumor when transplanted into a host. CSCs are correlated with poor clinical outcome due to their contribution to chemotherapy resistance and metastasis. Multiple cell surface and enzymatic markers have been characterized to identify CSCs within a heterogeneous tumor, and here we summarize ongoing preclinical and clinical efforts to therapeutically target these cells and improve patient outcomes. Stem Cells Translational Medicine 2019;8:75–81

Mlh1 deficiency increases the risk of hematopoietic malignancy after simulated space radiation exposure

Cancer-causing genome instability is a major concern during space travel due to exposure of astronauts to potent sources of high-linear energy transfer (LET) ionizing radiation. Hematopoietic stem cells (HSCs) are particularly susceptible to genotoxic stress, and accumulation of damage can lead to HSC dysfunction and oncogenesis. Our group recently demonstrated that aging human HSCs accumulate microsatellite instability coincident with loss of MLH1, a DNA Mismatch Repair (MMR) protein, which could reasonably predispose to radiation-induced HSC malignancies. Therefore, in an effort to reduce risk uncertainty for cancer development during deep space travel, we employed an Mlh1+/− mouse model to study the effects high-LET 56Fe ion space-like radiation. Irradiated Mlh1+/− mice showed a significantly higher incidence of lymphomagenesis with 56Fe ions compared to γ-rays and unirradiated mice, and malignancy correlated with increased MSI in the tumors. In addition, whole-exome sequencing analysis revealed high SNVs and INDELs in lymphomas being driven by loss of Mlh1 and frequently mutated genes had a strong correlation with human leukemias. Therefore, the data suggest that age-related MMR deficiencies could lead to HSC malignancies after space radiation, and that countermeasure strategies will be required to adequately protect the astronaut population on the journey to Mars.