Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery.

Rationale: High mortality in pancreatic cancer (PDAC) and triple negative breast cancer (TNBC) highlight the need to capitalize on nanoscale-design advantages for multifunctional diagnostics and therapies. DNA/RNA-therapies can provide potential breakthroughs, however, to date, there is no FDA-approved systemic delivery system to solid tumors. Methods: Here, we report a Janus-nanoparticle (jNP)-system with modular targeting, payload-delivery, and targeted-imaging capabilities. Our jNP-system consists of 10 nm ultrasmall superparamagnetic iron oxide nanoparticles (USPION) with opposing antibody-targeting and DNA/RNA payload-protecting faces, directionally self-assembled with commercially available zwitterionic microbubbles (MBs) and DNA/RNA payloads. Results: Sonoporation of targeted jNP-payload-MBs delivers functional reporter-DNA imparting tumor-fluorescence, and micro-RNA126 reducing non-druggable KRAS in PDAC-Panc1 and TNBC-MB231 xenografted tumors. The targeting jNP-system enhances ultrasound-imaging of intra-tumoral microvasculature using less MBs/body weight (BW). The jNP-design enhances USPION's T2*-magnetic resonance (MR) and MR-imaging of PDAC-peritoneal metastases using less Fe/BW. Conclusion: Altogether, data advance the asymmetric jNP-design as a potential theranostic Janus-USPION Modular Platform - a JUMP forward.

Humanized anti-DEspR IgG4S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis ratnu/nu model.

BACKGROUND: Pancreatic peritoneal carcinomatosis (PPC), with the worst median overall-survival (mOS), epitomizes the incurability of metastatic cancer. Cancer stem cells (CSCs) underpin this incurability. However, inhibitors of CSC-stemness fail to increase mOS in cancer patients despite preclinical tumor-reduction. This shortfall reinforces that preclinical efficacy should be defined by increased mOS in the presence of cancer comorbidities, CSC-heterogeneity and plasticity. The primary objectives of this study are: to test the dual endothelin-1/signal peptide receptor, DEspR, as a nodal therapeutic target in PPC, given DEspR induction in anoikis-resistant pancreatic CSCs, and to validate humanized anti-DEspR antibody, hu-6g8, as a potential therapeutic for PPC. METHODS: We used heterogeneous pools of CSCs selected for anoikis resistance from reprogrammed Panc1 and MiaPaCa2 tumor cells (TCs), and adherent TCs reprogrammed from CSCs (cscTCs). We used multiple anti-DEspR blocking antibodies (mAbs) with different epitopes, and a humanized anti-DEspR recombinant mAb cross-reactive in rodents and humans, to test DEspR inhibition effects. We measured DEspR-inhibition efficacy on multiple prometastatic CSC-functions in vitro, and on tumorigenesis and overall survival in a CSC-derived xenograft (CDX) nude rat model of PPC with comorbidities. RESULTS: Here we show that DEspR, a stress-survival receptor, is present on subsets of PDAC Panc1-TCs, TC-derived CSCs, and CSC-differentiated TCs (cscTCs), and that DESpR-inhibition decreases apoptosis-resistance and pro-metastatic mesenchymal functions of CSCs and cscTCs in vitro. We resolve the DNA-sequence/protein-function discordance by confirming ADAR1-RNA editing-dependent DEspR-protein expression in Panc1 and MiaPaCa2 TCs. To advance DEspR-inhibition as a nodal therapeutic approach for PPC, we developed and show improved functionality of a recombinant, humanized anti-DEspR IgG4S228P antibody, hu-6g8, over murine precursor anti-DEspR mabs. Hu-6g8 internalizes and translocates to the nucleus colocalized with cyto-nuclear shuttling galectins-1/3, and induces apoptotic cell changes. DEspR-inhibition blocks transperitoneal dissemination and progression to peritoneal carcinomatosis of heterogeneous DEspR±/CD133 ± Panc1-derived CSCs in xenografted nude rats, improving mOS without chemotherapy-like adverse effects. Lastly, we show DEspR expression in Stage II-IV primary and invasive TCs in the stroma in PDAC-patient tumor arrays. CONCLUSION: Collectively, the data support humanized anti-DEspR hu-6g8 as a potential targeted antibody-therapeutic with promising efficacy, safety and prevalence profiles for PPC patients.

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

BACKGROUND: In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. METHODS: To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. RESULTS: Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. CONCLUSIONS: Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

A functional 12T-insertion polymorphism in the ATP1A1 promoter confers decreased susceptibility to hypertension in a male Sardinian population.

Identification of susceptibility genes for essential hypertension in humans has been a challenge due to its multifactorial pathogenesis complicated by gene-gene and gene-environment interactions, developmental programing and sex specific differences. These concurrent features make identification of causal hypertension susceptibility genes with a single approach difficult, thus requiring multiple lines of evidence involving genetic, biochemical and biological experimentation to establish causal functional mutations. Here we report experimental evidence encompassing genetic, biochemical and in vivo modeling that altogether support ATP1A1 as a hypertension susceptibility gene in males in Sardinia, Italy. ATP1A1 encodes the α1Na,K-ATPase isoform, the sole sodium pump in vascular endothelial and renal tubular epithelial cells. DNA-sequencing detected a 12-nucleotide long thymidine (12T) insertion(ins)/deletion(del) polymorphism within a poly-T sequence (38T vs 26T) in the ATP1A1 5'-regulatory region associated with hypertension in a male Sardinian population. The 12T-insertion allele confers decreased susceptibility to hypertension (P = 0.035; OR = 0.50 [0.28-0.93]) accounting for 12.1 mmHg decrease in systolic BP (P = 0.02) and 6.6 mmHg in diastolic BP (P = 0.046). The ATP1A1 promoter containing the 12T-insertion exhibited decreased transcriptional activity in in vitro reporter-assay systems, indicating decreased α1Na,K-ATPase expression with the 12T-insertion, compared with the 12T-deletion ATP1A1 promoter. To test the effects of decreased α1Na,K-ATPase expression on blood pressure, we measured blood pressure by radiotelemetry in three month-old, highly inbred heterozygous knockout ATP1A1+/- male mice with resultant 58% reduction in ATP1A1 protein levels. Male ATP1A1+/- mice showed significantly lower blood pressure (P < 0.03) than age-matched male wild-type littermate controls. Concordantly, lower ATP1A1 expression is expected to lower Na-reabsorption in the kidney thereby decreasing sodium-associated risk for hypertension and sodium-induced endothelial stiffness and dysfunction. Altogether, data support ATP1A1 as a hypertension susceptibility gene in a male Sardinian population, and mandate further investigation of its involvement in hypertension in the general population.

Aortic and carotid arterial stiffness and epigenetic regulator gene expression changes precede blood pressure rise in stroke-prone Dahl salt-sensitive hypertensive rats.

Multiple clinical studies show that arterial stiffness, measured as pulse wave velocity (PWV), precedes hypertension and is an independent predictor of hypertension end organ diseases including stroke, cardiovascular disease and chronic kidney disease. Risk factor studies for arterial stiffness implicate age, hypertension and sodium. However, causal mechanisms linking risk factor to arterial stiffness remain to be elucidated. Here, we studied the causal relationship of arterial stiffness and hypertension in the Na-induced, stroke-prone Dahl salt-sensitive (S) hypertensive rat model, and analyzed putative molecular mechanisms. Stroke-prone and non-stroke-prone male and female rats were studied at 3- and 6-weeks of age for arterial stiffness (PWV, strain), blood pressure, vessel wall histology, and gene expression changes. Studies showed that increased left carotid and aortic arterial stiffness preceded hypertension, pulse pressure widening, and structural wall changes at the 6-week time-point. Instead, differential gene induction was detected implicating molecular-functional changes in extracellular matrix (ECM) structural constituents, modifiers, cell adhesion, and matricellular proteins, as well as in endothelial function, apoptosis balance, and epigenetic regulators. Immunostaining testing histone modifiers Ep300, HDAC3, and PRMT5 levels confirmed carotid artery-upregulation in all three layers: endothelial, smooth muscle and adventitial cells. Our study recapitulates observations in humans that given salt-sensitivity, increased Na-intake induced arterial stiffness before hypertension, increased pulse pressure, and structural vessel wall changes. Differential gene expression changes associated with arterial stiffness suggest a molecular mechanism linking sodium to full-vessel wall response affecting gene-networks involved in vascular ECM structure-function, apoptosis balance, and epigenetic regulation.

Sex-specific effects on spatial learning and memory, and sex-independent effects on blood pressure of a <3.3 Mbp rat chromosome 2 QTL region in Dahl salt-sensitive rats.

Epidemiological studies have consistently found that hypertension is associated with poor cognitive performance. We hypothesize that a putative causal mechanism underlying this association is due to genetic loci affecting both blood pressure and cognition. Consistent with this notion, we reported several blood pressure (BP) quantitative trait loci (QTLs) that co-localized with navigational performance (Nav)-QTLs influencing spatial learning and memory in Dahl rats. The present study investigates a chromosome 2 region harboring BP-f4 and Nav-8 QTLs. We developed two congenic strains, S.R2A and S.R2B introgressing Dahl R-chromosome 2 segments into Dahl S chromosome 2 region spanning BP-f4 and Nav-8 QTLs. Radiotelemetric blood pressure analysis identified only S.R2A congenic rats with lower systolic blood pressure (females: -26.0 mmHg, P = 0.003; males: -30.9 mmHg, P<1×10(-5)), diastolic blood pressure (females: -21.2 mmHg, P = 0.01; males: -25.7 mmHg, P<1×10(-5)), and mean arterial pressure (females: -23.9 mmHg, P = 0.004; males: -28.0 mmHg, P<1×10(-5)) compared with corresponding Dahl S controls, confirming the presence of BP-f4 QTL on rat chromosome 2. The S.R2B congenic segment did not affect blood pressure. Testing of S.R2A, S.R2B, and Dahl S male rats in the Morris water maze (MWM) task revealed significantly decreased spatial navigation performance in S.R2A male congenic rats when compared with Dahl S male controls (P<0.05). The S.R2B congenic segment did not affect performance of the MWM task in males. The S.R2A female rats did not differ in spatial navigation when compared with Dahl S female controls, indicating that the Nav-8 effect on spatial navigation is male-specific. Our results suggest the existence of a single QTL on chromosome 2 176.6-179.9 Mbp region which affects blood pressure in both males and females and cognition solely in males.

Analysis of CD45- [CD34+/KDR+] endothelial progenitor cells as juvenile protective factors in a rat model of ischemic-hemorrhagic stroke.

BACKGROUND: Identification of juvenile protective factors (JPFs) which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs) have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. A priori, if EPC aging plays a 'master-switch JPF-role' in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset. METHODOLOGY/PRINCIPAL FINDINGS: FACS analysis revealed that CD45- [CD34+/KDR+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR) and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[CD34+/KDR+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003). Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in CD45-[CD34+/KDR]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001), suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age-associated intimal hyperplasia. CONCLUSIONS/SIGNIFICANCE: Altogether, the data demonstrate that CD45-[CD34/KDR+]EPCs are juvenile protective factors for ischemic hemorrhagic stroke as modeled in the spTg25-rat model. The ability to delay stroke onset emphasizes the importance of EPC-mediated roles in vascular health for ischemic-hemorrhagic stroke, a high unmet need.

Worse renal disease in postmenopausal F2[Dahl S x R]-intercross rats: detection of novel QTLs affecting hypertensive kidney disease.

The prevalence of hypertension increases after menopause with 75% of postmenopausal women developing hypertension in the United States, along with hypertensive end organ diseases. While human and animal model studies have indicated a protective role for estrogen against cardiovascular disease and glomerulosclerosis, clinical studies of hormone replacement therapy in postmenopausal women have shown polar results with some improvement in hypertension but worsening of hypertensive kidney disease, or no effect at all. These observations suggest that the pathogenesis of postmenopausal hypertension and its target organ complications is more complex than projected, and that loss of endogenous estrogens induces epigenetic changes that alter genetic susceptibility to end-organ complications per se resulting in pathogenetic mechanisms beyond correction by hormone replacement. We studied postmenopausal-induced changes in renal disease and performed a total genome scan for quantitative trait loci (QTLs) affecting kidney disease in postmenopausal 16m-old F2[Dahl S x R]-intercross female rats. We used glomerular injury score (GIS) as quantitative trait. We compared QTLs amongst premenopausal, ovariectomized and postmenopausal F2[Dahl S x R]-intercross rats using identical phenotype characterization. Postmenopausal F2[Dahl S x R]-intercross rats exhibited increased hypertensive glomerulosclerosis (P<0.01) and equivalent levels of kidney disease when compared to premenopausal and ovariectomized F2[Dahl S x R]-intercross rats respectively. We detected three significant to highly significant GIS-QTLs (GIS-pm1 on chromosome 4, LOD 3.54; GIS-pm2 on chromosome 3, LOD 2.72; GIS-pm3 on chromosome 5, LOD 2.37) and two suggestive GIS-QTLs (GIS-pm4 on chromosome 2, LOD 1.70; GIS-pm5 on chromosome 7, LOD 1.28), all of which were unique to this postmenopausal population. Detection of increased renal disease phenotype in postmenopausal and ovariectomized subjects suggests a protective role of ovarian hormones. Furthermore, the detection of distinct GIS-QTLs in postmenopausal intercross female rats suggests that distinct genetic mechanisms underlie hypertensive glomerulosclerosis in premenopausal and postmenopausal states.

Dahl (S x R) congenic strain analysis confirms and defines a chromosome 5 female-specific blood pressure quantitative trait locus to <7 Mbp.

The detection of multiple sex-specific blood pressure (BP) quantitative trait loci (QTLs) in independent total genome analyses of F2 (Dahl S x R)-intercross male and female rat cohorts confirms clinical observations of sex-specific disease cause and response to treatment among hypertensive patients, and mandate the identification of sex-specific hypertension genes/mechanisms. We developed and studied two congenic strains, S.R5A and S.R5B introgressing Dahl R-chromosome 5 segments into Dahl S chromosome 5 region spanning putative BP-f1 and BP-f2 QTLs. Radiotelemetric non-stressed 24-hour BP analysis at four weeks post-high salt diet (8% NaCl) challenge, identified only S.R5B congenic rats with lower SBP (-26.5 mmHg, P = 0.002), DBP (-23.7 mmHg, P = 0.004) and MAP (-25.1 mmHg, P = 0.002) compared with Dahl S female controls at four months of age confirming BP-f1 but not BP-f2 QTL on rat chromosome 5. The S.R5B congenic segment did not affect pulse pressure and relative heart weight indicating that the gene underlying BP-f1 does not influence arterial stiffness and cardiac hypertrophy. The results of our congenic analysis narrowed BP-f1 to chromosome 5 coordinates 134.9-141.5 Mbp setting up the basis for further fine mapping of BP-f1 and eventual identification of the specific gene variant accounting for BP-f1 effect on blood pressure.

Differential genetic basis for pre-menopausal and post-menopausal salt-sensitive hypertension.

Essential hypertension affects 75% of post-menopausal women in the United States causing greater cardiovascular complications compared with age-matched men and pre-menopausal women. Hormone replacement and current anti-hypertensive therapies do not correct this post-menopausal increased risk suggesting a distinct pathogenic framework. We investigated the hypothesis that distinct genetic determinants might underlie susceptibility to salt sensitive hypertension in pre-menopausal and post-menopausal states. To determine whether distinct genetic loci contribute to post-menopausal salt-sensitive hypertension, we performed a genome-wide scan for quantitative trait loci (QTLs) affecting blood pressure (BP) in 16-month old post-menopausal F2 (Dahl S×R)-intercross female rats characterized for blood pressure by radiotelemetry. Given identical environments and high salt challenge, post-menopausal BP levels were significantly higher than observed in pre-menopausal (post-menopausal versus pre-menopausal SBP, P<0.0001) and ovariectomized (post-menopausal versus ovariectomized SBP, P<0.001) F2-intercross female rats. We detected four significant to highly significant BP-QTLs (BP-pm1 on chromosome 13, LOD 3.78; BP-pm2 on chromosome 11, LOD 2.76; BP-pm3 on chromosome 2, LOD 2.61; BP-pm4 on chromosome 4, LOD 2.50) and two suggestive BP-QTLs (BP-pm5 on chromosome 15, LOD 2.37; BP-f1 on chromosome 5, LOD 1.65), four of which (BP-pm2, BP-pm3, BP-pm4, BP-pm5) were unique to this post-menopausal cohort. These data demonstrate distinct polygenic susceptibility underlying post-menopausal salt-sensitive hypertension providing a pathway towards the identification of mechanism-based therapy for post-menopausal hypertension and ensuing target-organ complications.

Prestroke proteomic changes in cerebral microvessels in stroke-prone, transgenic[hCETP]-Hyperlipidemic, Dahl salt-sensitive hypertensive rats.

Stroke is the third leading cause of death in the United States with high rates of morbidity among survivors. The search to fill the unequivocal need for new therapeutic approaches would benefit from unbiased proteomic analyses of animal models of spontaneous stroke in the prestroke stage. Since brain microvessels play key roles in neurovascular coupling, we investigated prestroke microvascular proteome changes. Proteomic analysis of cerebral cortical microvessels (cMVs) was done by tandem mass spectrometry comparing two prestroke time points. Metaprotein-pathway analyses of proteomic spectral count data were done to identify risk factor-induced changes, followed by QSPEC-analyses of individual protein changes associated with increased stroke susceptibility. We report 26 cMV proteome profiles from male and female stroke-prone and non-stroke-prone rats at 2 months and 4.5 months of age prior to overt stroke events. We identified 1,934 proteins by two or more peptides. Metaprotein pathway analysis detected age-associated changes in energy metabolism and cell-to-microenvironment interactions, as well as sex-specific changes in energy metabolism and endothelial leukocyte transmigration pathways. Stroke susceptibility was associated independently with multiple protein changes associated with ischemia, angiogenesis or involved in blood brain barrier (BBB) integrity. Immunohistochemical analysis confirmed aquaporin-4 and laminin-α1 induction in cMVs, representative of proteomic changes with >65 Bayes factor (BF), associated with stroke susceptibility. Altogether, proteomic analysis demonstrates significant molecular changes in ischemic cerebral microvasculature in the prestroke stage, which could contribute to the observed model phenotype of microhemorrhages and postischemic hemorrhagic transformation. These pathways comprise putative targets for translational research of much needed novel diagnostic and therapeutic approaches for stroke.

Successful outcome with early renal transplantation following treatment of encapsulating peritoneal sclerosis.

Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis (PD) associated with cocooning of the bowel within a sheath of fibrous tissue causing obstruction. Uncertainty remains regarding the aetiology, pathogenesis and optimal treatment. The current management of EPS is largely based on case reports and small series, and there are limited data regarding outcomes of patients who have undergone renal transplantation following treatment for EPS. We present the case of a 28-year-old man with EPS who was treated surgically followed by anti-fibrotic therapy, with tamoxifen, and 7 months later proceeded to renal transplantation with good outcome.