Amar J. Majmundar

Hypoxia inducible factors and the response to hypoxic stress

Oxygen (O(2)) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O(2) availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors, including factor-inhibiting HIF1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway.

HIF2α inhibition promotes p53 pathway activity, tumor cell death, and radiation responses

Approximately 50% of cancer patients receive radiation treatment, either alone or in combination with other therapies. Tumor hypoxia has long been associated with resistance to radiation therapy. Moreover, the expression of hypoxia inducible factors HIF1α and/or HIF2α correlates with poor prognosis in many tumors. Recent evidence indicates that HIF1α expression can enhance radiation-induced apoptosis in cancer cells. We demonstrate here that HIF2α inhibition promotes tumor cell death and, in contrast to HIF1α, enhances the response to radiation treatment. Specifically, inhibiting HIF2α expression augments p53 activity, increases apoptosis, and reduces clonogenic survival of irradiated and non-irradiated cells. Moreover, HIF2α inhibition promotes p53-mediated responses by disrupting cellular redox homeostasis, thereby permitting reactive oxygen species (ROS) accumulation and DNA damage. These results correlate with altered p53 phosphorylation and target gene expression in untreated human tumor samples and show that HIF2α likely contributes to tumor cell survival including during radiation therapy.

Endothelial HIF-2α regulates murine pathological angiogenesis and revascularization processes

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.

O2 Regulates Skeletal Muscle Progenitor Differentiation through Phosphatidylinositol 3-Kinase/AKT Signaling

ABSTRACT Skeletal muscle stem/progenitor cells, which give rise to terminally differentiated muscle, represent potential therapies for skeletal muscle diseases. Delineating the factors regulating these precursors will facilitate their reliable application in human muscle repair. During embryonic development and adult regeneration, skeletal muscle progenitors reside in low-O2 environments before local blood vessels and differentiated muscle form. Prior studies established that low O2 levels (hypoxia) maintained muscle progenitors in an undifferentiated state in vitro, although it remained unclear if progenitor differentiation was coordinated with O2 availability in vivo. In addition, the molecular signals linking O2 to progenitor differentiation are incompletely understood. Here we show that the muscle differentiation program is repressed by hypoxia in vitro and ischemia in vivo. Surprisingly, hypoxia can significantly impair differentiation in the absence of hypoxia-inducible factors (HIFs), the primary developmental effectors of O2. In order to maintain the undifferentiated state, low O2 levels block the phosphatidylinositol 3-kinase/AKT pathway in a predominantly HIF1α-independent fashion. O2 deprivation affects AKT activity by reducing insulin-like growth factor I receptor sensitivity to growth factors. We conclude that AKT represents a key molecular link between O2 and skeletal muscle differentiation.

Hemodynamic and metabolic diffuse optical monitoring in a mouse model of hindlimb ischemia

Murine hindlimb ischemia is a useful model for investigation of the mechanisms of peripheral arterial disease and for understanding the role of endothelial cells and generic factors affecting vascular regeneration or angiogenesis. To date, important research with these models has explored tissue reperfusion following ischemia with Laser Doppler methods, methods which provide information about superficial (~mm) vascular regeneration. In this work, we employ diffuse correlation spectroscopy (DCS) and diffuse optical spectroscopy (DOS) in mice after hindlimb ischemia. We hypothesize that vascular re-growth is not uniform in tissue, and therefore, since diffuse optical methods are capable of probing deep tissues, that the diffuse optics approach will provide a more complete picture of the angiogenesis process throughout the whole depth profile of the limb. Besides increased depth penetration, the combined measurements of DCS and DOS enable all-optical, noninvasive, longitudinal monitoring of tissue perfusion and oxygenation that reveals the interplay between these hemodynamic parameters during angiogenesis. Control mice were found to reestablish 90% of perfusion and oxygen consumption during this period, but oxygen saturation in the limb only partially recovered to about 30% of its initial value. The vascular recovery of mice with endothelial cell-specific deletion of HIF-2α was found to be significantly impaired relative to control mice, indicating that HIF-2α is important for endothelial cell functions in angiogenesis. Comparison of DOS/DCS measurements to parallel measurements in the murine models using Laser Doppler Flowmetry reveal differences in the reperfusion achieved by superficial versus deep tissue during neoangiogenesis; findings from histological analysis of blood vessel development were further correlated with these differences. In general, the combination of DCS and DOS enables experimenters to obtain useful information about oxygenation, metabolism, and perfusion throughout the limb. The results establish diffuse optics as a practical noninvasive method to evaluate the role of transcription factors, such as the endothelial cell-specific HIF-2α, in genetic ally modified mice.

Epigenetic downregulation of the DNA repair gene MED1/MBD4 in colorectal and ovarian cancer.

MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated (≥ 50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p

Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome

BACKGROUND AND OBJECTIVES Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. RESULTS In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. CONCLUSIONS Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.

Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.Nephrotic syndrome is the second most common chronic kidney disease but there are no targeted treatment strategies available. Here the authors identify mutations of six genes codifying for proteins involved in cytoskeleton remodelling and modulation of small GTPases in 17 families with nephrotic syndrome.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.