Approximation of the Ankle-Brachial Index in the Setting of Medial Arterial Calcific Sclerosis.

The presence of medial arterial calcific sclerosis is known to cause inaccuracy in the interpretation of noninvasive vascular testing. This substantially limits the utility of an important baseline diagnostic test for peripheral arterial disease. Therefore, the objective of this investigation was to derive a method to effectively factor out calcification in the interpretation of the ankle and digital brachial indices. The noninvasive vascular testing results of 160 subjects were stratified into the absence of calcification, mild calcification, moderate calcification, and severe calcification based on plain film radiographic findings of the infrageniculate vessels. Measurements were then performed of the pulse volume recording (PVR) waveforms at brachial, ankle and digital anatomic levels to include PVR wavelength and PVR upstroke length, with a calculation of the ratio of PVR upstroke length to PVR wavelength. These measurements were compared between groups and then correlated to the ankle and digital brachial indices. A significant difference was observed in the PVR upstroke ratio between the 3 anatomic levels (0.1818 vs 0.2622 vs 0.3191; p < .001), but not between the 4 calcification groups (0.2457 vs 0.2363 vs 0.2694 vs 0.2631; p = .242). A significant negative correlation was observed between the PVR upstroke ratio and the ankle brachial index (ABI) (Pearson -0.454; p = .002) with linear regression indicating the relationship is defined by the formula: Effective ankle brachial index = 1.17 - (1.33 × PVR upstroke ratio at ankle level). A significant negative correlation was also observed between the PVR upstroke ratio and the digital brachial index (Pearson -0.553; p < .001) with linear regression indicating the relationship is defined by the formula: Effective toe brachial index = 1.04 - (1.61 × PVR upstroke ratio at digital level). The results of this investigation demonstrate the feasibility of, and provide equations to approximate, the effective ankle brachial and toe brachial indices in the setting of medial arterial calcification.

Responsiveness and Inter-Rater Reliability of the Pulse Volume Recording Upstroke Ratio (PVRr).

The objective of this study was to evaluate a measure of the responsiveness and reliability of the pulse volume recording upstroke ratio (PVRr). A database of 389 subjects undergoing lower extremity revascularization was analyzed. Subjects were included in the analysis if they had undergone pedal radiographs, had PVRs performed pre- and postlower extremity revascularization, and had regular pulsatile digital waveforms with a pressure recording on both PVRs. The responsiveness of the PVRr was assessed by means of the postoperative percent change in comparison to the digital pressures. A statistically significant negative correlation was observed (Pearson -0.421; p = .007) indicating that as digital pressures increased, the PVRr decreased. Further, measurement of the reliability of the PVRr was performed on a selection of 10 recordings by 2 residents and 3 board-certified surgeons. The observed intraclass correlation coefficient of measurements was 0.960. Results of this investigation provide evidence in support of the responsiveness and inter-rater reliability in the calculation of the pulse volume recording upstroke ratio.

Novel Knowledge-Based Transcriptomic Profiling of Lipid Lysophosphatidylinositol-Induced Endothelial Cell Activation.

To determine whether pro-inflammatory lipid lysophosphatidylinositols (LPIs) upregulate the expressions of membrane proteins for adhesion/signaling and secretory proteins in human aortic endothelial cell (HAEC) activation, we developed an EC biology knowledge-based transcriptomic formula to profile RNA-Seq data panoramically. We made the following primary findings: first, G protein-coupled receptor 55 (GPR55), the LPI receptor, is expressed in the endothelium of both human and mouse aortas, and is significantly upregulated in hyperlipidemia; second, LPIs upregulate 43 clusters of differentiation (CD) in HAECs, promoting EC activation, innate immune trans-differentiation, and immune/inflammatory responses; 72.1% of LPI-upregulated CDs are not induced in influenza virus-, MERS-CoV virus- and herpes virus-infected human endothelial cells, which hinted the specificity of LPIs in HAEC activation; third, LPIs upregulate six types of 640 secretomic genes (SGs), namely, 216 canonical SGs, 60 caspase-1-gasdermin D (GSDMD) SGs, 117 caspase-4/11-GSDMD SGs, 40 exosome SGs, 179 Human Protein Atlas (HPA)-cytokines, and 28 HPA-chemokines, which make HAECs a large secretory organ for inflammation/immune responses and other functions; fourth, LPIs activate transcriptomic remodeling by upregulating 172 transcription factors (TFs), namely, pro-inflammatory factors NR4A3, FOS, KLF3, and HIF1A; fifth, LPIs upregulate 152 nuclear DNA-encoded mitochondrial (mitoCarta) genes, which alter mitochondrial mechanisms and functions, such as mitochondrial organization, respiration, translation, and transport; sixth, LPIs activate reactive oxygen species (ROS) mechanism by upregulating 18 ROS regulators; finally, utilizing the Cytoscape software, we found that three mechanisms, namely, LPI-upregulated TFs, mitoCarta genes, and ROS regulators, are integrated to promote HAEC activation. Our results provide novel insights into aortic EC activation, formulate an EC biology knowledge-based transcriptomic profile strategy, and identify new targets for the development of therapeutics for cardiovascular diseases, inflammatory conditions, immune diseases, organ transplantation, aging, and cancers.

Reply to Comment on Shen H, et al. "Co-signaling receptors regulate T-cell plasticity and immune tolerance". Frontiers in Bioscience-Landmark. 2019; 24: 96-132.

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Procaspase-1 patrolled to the nucleus of proatherogenic lipid LPC-activated human aortic endothelial cells induces ROS promoter CYP1B1 and strong inflammation.

To determine the roles of nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs) activated by proatherogenic lipid lysophosphatidylcholine (LPC), we examined cytosolic and nuclear localization of pro-caspase-1, identified nuclear export signal (NES) in pro-caspase-1 and sequenced RNAs. We made the following findings: 1) LPC increases nuclear localization of procaspase-1 in HAECs. 2) Nuclear pro-caspase-1 exports back to the cytosol, which is facilitated by a leptomycin B-inhibited mechanism. 3) Increased nuclear localization of pro-caspase-1 by a new NES peptide inhibitor upregulates inflammatory genes in oxidative stress and Th17 pathways; and SUMO activator N106 enhances nuclear localization of pro-caspase-1 and caspase-1 activation (p20) in the nucleus. 4) LPC plus caspase-1 enzymatic inhibitor upregulates inflammatory genes with hypercytokinemia/hyperchemokinemia and interferon pathways, suggesting a novel capsase-1 enzyme-independent inflammatory mechanism. 5) LPC in combination with NES inhibitor and caspase-1 inhibitor upregulate inflammatory gene expression that regulate Th17 activation, endotheli-1 signaling, p38-, and ERK- MAPK pathways. To examine two hallmarks of endothelial activation such as secretomes and membrane protein signaling, LPC plus NES inhibitor upregulate 57 canonical secretomic genes and 76 exosome secretomic genes, respectively, promoting four pathways including Th17, IL-17 promoted cytokines, interferon signaling and cholesterol biosynthesis. LPC with NES inhibitor also promote inflammation via upregulating ROS promoter CYP1B1 and 11 clusters of differentiation (CD) membrane protein pathways. Mechanistically, all the LPC plus NES inhibitor-induced genes are significantly downregulated in CYP1B1-deficient microarray, suggesting that nuclear caspase-1-induced CYP1B1 promotes strong inflammation. These transcriptomic results provide novel insights on the roles of nuclear caspase-1 in sensing DAMPs, inducing ROS promoter CYP1B1 and in regulating a large number of genes that mediate HAEC activation and inflammation. These findings will lead to future development of novel therapeutics for cardiovascular diseases (CVD), inflammations, infections, transplantation, autoimmune disease and cancers. (total words: 284).

Endothelial Immunity Trained by Coronavirus Infections, DAMP Stimulations and Regulated by Anti-Oxidant NRF2 May Contribute to Inflammations, Myelopoiesis, COVID-19 Cytokine Storms and Thromboembolism.

To characterize transcriptomic changes in endothelial cells (ECs) infected by coronaviruses, and stimulated by DAMPs, the expressions of 1311 innate immune regulatomic genes (IGs) were examined in 28 EC microarray datasets with 7 monocyte datasets as controls. We made the following findings: The majority of IGs are upregulated in the first 12 hours post-infection (PI), and maintained until 48 hours PI in human microvascular EC infected by middle east respiratory syndrome-coronavirus (MERS-CoV) (an EC model for COVID-19). The expressions of IGs are modulated in 21 human EC transcriptomic datasets by various PAMPs/DAMPs, including LPS, LPC, shear stress, hyperlipidemia and oxLDL. Upregulation of many IGs such as nucleic acid sensors are shared between ECs infected by MERS-CoV and those stimulated by PAMPs and DAMPs. Human heart EC and mouse aortic EC express all four types of coronavirus receptors such as ANPEP, CEACAM1, ACE2, DPP4 and virus entry facilitator TMPRSS2 (heart EC); most of coronavirus replication-transcription protein complexes are expressed in HMEC, which contribute to viremia, thromboembolism, and cardiovascular comorbidities of COVID-19. ECs have novel trained immunity (TI), in which subsequent inflammation is enhanced. Upregulated proinflammatory cytokines such as TNFα, IL6, CSF1 and CSF3 and TI marker IL-32 as well as TI metabolic enzymes and epigenetic enzymes indicate TI function in HMEC infected by MERS-CoV, which may drive cytokine storms. Upregulated CSF1 and CSF3 demonstrate a novel function of ECs in promoting myelopoiesis. Mechanistically, the ER stress and ROS, together with decreased mitochondrial OXPHOS complexes, facilitate a proinflammatory response and TI. Additionally, an increase of the regulators of mitotic catastrophe cell death, apoptosis, ferroptosis, inflammasomes-driven pyroptosis in ECs infected with MERS-CoV and the upregulation of pro-thrombogenic factors increase thromboembolism potential. Finally, NRF2-suppressed ROS regulate innate immune responses, TI, thrombosis, EC inflammation and death. These transcriptomic results provide novel insights on the roles of ECs in coronavirus infections such as COVID-19, cardiovascular diseases (CVD), inflammation, transplantation, autoimmune disease and cancers.

Canonical Secretomes, Innate Immune Caspase-1-, 4/11-Gasdermin D Non-Canonical Secretomes and Exosomes May Contribute to Maintain Treg-Ness for Treg Immunosuppression, Tissue Repair and Modulate Anti-Tumor Immunity via ROS Pathways.

We performed a transcriptomic analyses using the strategies we pioneered and made the following findings: 1) Normal lymphoid Tregs, diseased kidney Tregs, splenic Tregs from mice with injured muscle have 3, 17 and 3 specific (S-) pathways, respectively; 2) Tumor splenic Tregs share 12 pathways with tumor Tregs; tumor splenic Tregs and tumor Tregs have 11 and 8 S-pathways, respectively; 3) Normal and non-tumor disease Tregs upregulate some of novel 2641 canonical secretomic genes (SGs) with 24 pathways, and tumor Tregs upregulate canonical secretomes with 17 pathways; 4) Normal and non-tumor disease tissue Tregs upregulate some of novel 6560 exosome SGs with 56 exosome SG pathways (ESP), tumor Treg ESP are more focused than other Tregs; 5) Normal, non-tumor diseased Treg and tumor Tregs upregulate some of novel 961 innate immune caspase-1 SGs and 1223 innate immune caspase-4 SGs to fulfill their tissue/SG-specific and shared functions; 6) Most tissue Treg transcriptomes are controlled by Foxp3; and Tumor Tregs had increased Foxp3 non-collaboration genes with ROS and 17 other pathways; 7) Immune checkpoint receptor PD-1 does, but CTLA-4 does not, play significant roles in promoting Treg upregulated genes in normal and non-tumor disease tissue Tregs; and tumor splenic and tumor Tregs have certain CTLA-4-, and PD-1-, non-collaboration transcriptomic changes with innate immune dominant pathways; 8) Tumor Tregs downregulate more immunometabolic and innate immune memory (trained immunity) genes than Tregs from other groups; and 11) ROS significantly regulate Treg transcriptomes; and ROS-suppressed genes are downregulated more in tumor Tregs than Tregs from other groups. Our results have provided novel insights on the roles of Tregs in normal, injuries, regeneration, tumor conditions and some of canonical and innate immune non-canonical secretomes via ROS-regulatory mechanisms and new therapeutic targets for immunosuppression, tissue repair, cardiovascular diseases, chronic kidney disease, autoimmune diseases, transplantation, and cancers.

Level of Agreement Between Systematic Doppler Examination of the Lower Extremity and Diagnostic Angiography in the Setting of Peripheral Arterial Disease.

BACKGROUND: The objective of this investigation was to determine the level of agreement between a systematic clinical Doppler examination of the foot and ankle and diagnostic peripheral angiography. METHODS: The described Doppler examination technique attempted to determine the patency, quality, and direction of the flow through the dorsalis pedis artery, posterior tibial artery, terminal branches of the peroneal artery, and vascular arch of the foot. These results were then compared with angiographic distal run-off images as interpreted by a blinded vascular surgeon. RESULTS: Levels of agreement with respect to artery patency/quality ranged from 64.0% to 84.0%. Sensitivity ranged from 53.8% to 84.2%, and specificity ranged from 64.7% to 91.7%. Agreement with respect to arterial flow direction ranged from 73.3% to 90.5%. CONCLUSIONS: We interpret these results to indicate that this comprehensive physical examination technique of the arterial flow to the foot and ankle with a Doppler device might serve as a reasonable initial surrogate to diagnostic angiography in some patients with peripheral arterial disease.

Evaluating the Impact of Medial Arterial Calcification on Outcomes of Infrageniculate Endovascular Interventions for Treatment of Diabetic Foot Ulcers.

BACKGROUND: Medial arterial calcification (MAC) of the tibial and pedal arteries has been associated with an increased risk of amputation among people with diabetes. Endovascular interventions on infrageniculate vessels are frequently performed with the intent of treating peripheral artery disease (PAD) and decreasing the risk of amputation in those with diabetes. This study aimed to investigate how the extent of MAC impacts outcomes of endovascular procedures in people with diabetic foot ulcers (DFU). METHODS: We identified all patients who had undergone infrageniculate angioplasty in the setting of DFU at our institution between 2009 and 2019. Subjects were assigned a MAC score based on the severity of MAC in each vessel visualized on plain radiographs of the ankle and foot. We evaluated the relationship between MAC and the primary outcome, major adverse limb event (MALE), using stratified Cox proportional modeling. RESULTS: Among 99 subjects with DFU who had undergone infrageniculate angioplasty, MALE occurred in 50% (95% confidence interval [CI] 38%-61%) of patients within 1 year of intervention. On univariate Cox regression analysis, each 1 point increment in MAC score (hazard ratio [HR], 1.09; 95% CI 1.01-1.18), the third tertile of MAC score (HR, 2.27; 95% CI 1.01-5.11), age (HR 0.96; 95% CI 0.93-0.99), and wound grade (HR, 5.34; 95% CI 2.17-13.14), were significantly associated with increased risk of MALE. On adjusted analysis stratified by wound grade, MAC score was found to be associated with MALE only in patients with a low wound grade. CONCLUSION: Increased severity of MAC is associated with increased risk of MALE for subjects undergoing infrageniculate angioplasty with a low wound grade. Further research is needed to better understand the complex relationships of MAC, PAD, DFU, and interventions aimed at promoting healing of DFU.

Incidence of venous thromboembolism in coronavirus disease 2019: An experience from a single large academic center.

BACKGROUND: Infection with the novel severe acute respiratory syndrome coronavirus 2 has been associated with a hypercoagulable state. Emerging data from China and Europe have consistently shown an increased incidence of venous thromboembolism (VTE). We aimed to identify the VTE incidence and early predictors of VTE at our high-volume tertiary care center. METHODS: We performed a retrospective cohort study of 147 patients who had been admitted to Temple University Hospital with coronavirus disease 2019 (COVID-19) from April 1, 2020 to April 27, 2020. We first identified the VTE (pulmonary embolism [PE] and deep vein thrombosis [DVT]) incidence in our cohort. The VTE and no-VTE groups were compared by univariable analysis for demographics, comorbidities, laboratory data, and treatment outcomes. Subsequently, multivariable logistic regression analysis was performed to identify the early predictors of VTE. RESULTS: The 147 patients (20.9% of all admissions) admitted to a designated COVID-19 unit at Temple University Hospital with a high clinical suspicion of acute VTE had undergone testing for VTE using computed tomography pulmonary angiography and/or extremity venous duplex ultrasonography. The overall incidence of VTE was 17% (25 of 147). Of the 25 patients, 16 had had acute PE, 14 had had acute DVT, and 5 had had both PE and DVT. The need for invasive mechanical ventilation (adjusted odds ratio, 3.19; 95% confidence interval, 1.07-9.55) and the admission D-dimer level ≥1500 ng/mL (adjusted odds ratio, 3.55; 95% confidence interval, 1.29-9.78) were independent markers associated with VTE. The all-cause mortality in the VTE group was greater than that in the non-VTE group (48% vs 22%; P = .007). CONCLUSIONS: Our study represents one of the earliest reported from the United States on the incidence rate of VTE in patients with COVID-19. Patients with a high clinical suspicion and the identified risk factors (invasive mechanical ventilation, admission D-dimer level ≥1500 ng/mL) should be considered for early VTE testing. We did not screen all patients admitted for VTE; therefore, the true incidence of VTE could have been underestimated. Our findings require confirmation in future prospective studies.

Targeting mitochondrial fission as a potential therapeutic for abdominal aortic aneurysm.

AIMS: Angiotensin II (AngII) is a potential contributor to the development of abdominal aortic aneurysm (AAA). In aortic vascular smooth muscle cells (VSMCs), exposure to AngII induces mitochondrial fission via dynamin-related protein 1 (Drp1). However, pathophysiological relevance of mitochondrial morphology in AngII-associated AAA remains unexplored. Here, we tested the hypothesis that mitochondrial fission is involved in the development of AAA. METHODS AND RESULTS: Immunohistochemistry was performed on human AAA samples and revealed enhanced expression of Drp1. In C57BL6 mice treated with AngII plus ß-aminopropionitrile, AAA tissue also showed an increase in Drp1 expression. A mitochondrial fission inhibitor, mdivi1, attenuated AAA size, associated aortic pathology, Drp1 protein induction, and mitochondrial fission but not hypertension in these mice. Moreover, western-blot analysis showed that induction of matrix metalloproteinase-2, which precedes the development of AAA, was blocked by mdivi1. Mdivi1 also reduced the development of AAA in apolipoprotein E-deficient mice infused with AngII. As with mdivi1, Drp1+/- mice treated with AngII plus ß-aminopropionitrile showed a decrease in AAA compared to control Drp1+/+ mice. In abdominal aortic VSMCs, AngII induced phosphorylation of Drp1 and mitochondrial fission, the latter of which was attenuated with Drp1 silencing as well as mdivi1. AngII also induced vascular cell adhesion molecule-1 expression and enhanced leucocyte adhesion and mitochondrial oxygen consumption in smooth muscle cells, which were attenuated with mdivi1. CONCLUSION: These data indicate that Drp1 and mitochondrial fission play salient roles in AAA development, which likely involves mitochondrial dysfunction and inflammatory activation of VSMCs.

End-stage renal disease is different from chronic kidney disease in upregulating ROS-modulated proinflammatory secretome in PBMCs - A novel multiple-hit model for disease progression.

BACKGROUND: The molecular mechanisms underlying chronic kidney disease (CKD) transition to end-stage renal disease (ESRD) and CKD acceleration of cardiovascular and other tissue inflammations remain poorly determined. METHODS: We conducted a comprehensive data analyses on 7 microarray datasets in peripheral blood mononuclear cells (PBMCs) from patients with CKD and ESRD from NCBI-GEO databases, where we examined the expressions of 2641 secretome genes (SG). RESULTS: 1) 86.7% middle class (molecular weight >500 Daltons) uremic toxins (UTs) were encoded by SGs; 2) Upregulation of SGs in PBMCs in patients with ESRD (121 SGs) were significantly higher than that of CKD (44 SGs); 3) Transcriptomic analyses of PBMC secretome had advantages to identify more comprehensive secretome than conventional secretomic analyses; 4) ESRD-induced SGs had strong proinflammatory pathways; 5) Proinflammatory cytokines-based UTs such as IL-1ß and IL-18 promoted ESRD modulation of SGs; 6) ESRD-upregulated co-stimulation receptors CD48 and CD58 increased secretomic upregulation in the PBMCs, which were magnified enormously in tissues; 7) M1-, and M2-macrophage polarization signals contributed to ESRD- and CKD-upregulated SGs; 8) ESRD- and CKD-upregulated SGs contained senescence-promoting regulators by upregulating proinflammatory IGFBP7 and downregulating anti-inflammatory TGF-ß1 and telomere stabilizer SERPINE1/PAI-1; 9) ROS pathways played bigger roles in mediating ESRD-upregulated SGs (11.6%) than that in CKD-upregulated SGs (6.8%), and half of ESRD-upregulated SGs were ROS-independent. CONCLUSIONS: Our analysis suggests novel secretomic upregulation in PBMCs of patients with CKD and ESRD, act synergistically with uremic toxins, to promote inflammation and potential disease progression. Our findings have provided novel insights on PBMC secretome upregulation to promote disease progression and may lead to the identification of new therapeutic targets for novel regimens for CKD, ESRD and their accelerated cardiovascular disease, other inflammations and cancers. (Total words: 279).

A Novel Subset of CD95+ Pro-Inflammatory Macrophages Overcome miR155 Deficiency and May Serve as a Switch From Metabolically Healthy Obesity to Metabolically Unhealthy Obesity.

Metabolically healthy obesity (MHO) accounts for roughly 35% of all obese patients. There is no clear consensus that has been reached on whether MHO is a stable condition or merely a transitory period between metabolically healthy lean and metabolically unhealthy obesity (MUO). Additionally, the mechanisms underlying MHO and any transition to MUO are not clear. Macrophages are the most common immune cells in adipose tissues and have a significant presence in atherosclerosis. Fas (or CD95), which is highly expressed on macrophages, is classically recognized as a pro-apoptotic cell surface receptor. However, Fas also plays a significant role as a pro-inflammatory molecule. Previously, we established a mouse model (ApoE-/-/miR155-/-; DKO mouse) of MHO, based on the criteria of not having metabolic syndrome (MetS) and insulin resistance (IR). In our current study, we hypothesized that MHO is a transition phase toward MUO, and that inflammation driven by our newly classified CD95+CD86- macrophages is a novel mechanism for this transition. We found that, with extended (24 weeks) high-fat diet feeding (HFD), MHO mice became MUO, shown by increased atherosclerosis. Mechanistically, we found the following: 1) at the MHO stage, DKO mice exhibited increased pro-inflammatory markers in adipose tissue, including CD95, and serum; 2) total adipose tissue macrophages (ATMs) increased; 3) CD95+CD86- subset of ATMs also increased; and 4) human aortic endothelial cells (HAECs) were activated (as determined by upregulated ICAM1 expression) when incubated with conditioned media from CD95+-containing DKO ATMs and human peripheral blood mononuclear cells-derived macrophages in comparison to respective controls. These results suggest that extended HFD in MHO mice promotes vascular inflammation and atherosclerosis via increasing CD95+ pro-inflammatory ATMs. In conclusion, we have identified a novel molecular mechanism underlying MHO transition to MUO with HFD. We have also found a previously unappreciated role of CD95+ macrophages as a potentially novel subset that may be utilized to assess pro-inflammatory characteristics of macrophages, specifically in adipose tissue in the absence of pro-inflammatory miR-155. These findings have provided novel insights on MHO transition to MUO and new therapeutic targets for the future treatment of MUO, MetS, other obese diseases, and type II diabetes.

Homocysteine-methionine cycle is a metabolic sensor system controlling methylation-regulated pathological signaling.

Homocysteine-Methionine (HM) cycle produces universal methyl group donor S-adenosylmethione (SAM), methyltransferase inhibitor S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hyperhomocysteinemia (HHcy) is established as an independent risk factor for cardiovascular disease (CVD) and other degenerative disease. We selected 115 genes in the extended HM cycle (31 metabolic enzymes and 84 methyltransferases), examined their protein subcellular location/partner protein, investigated their mRNA levels and mapped their corresponding histone methylation status in 35 disease conditions via mining a set of public databases and intensive literature research. We have 6 major findings. 1) All HM metabolic enzymes are located only in the cytosol except for cystathionine-ß-synthase (CBS), which was identified in both cytosol and nucleus. 2) Eight disease conditions encountered only histone hypomethylation on 8 histone residues (H3R2/K4/R8/K9/K27/K36/K79 and H4R3). Nine disease conditions had only histone hypermethylation on 8 histone residues (H3R2/K4/K9/K27/K36/K79 and H4R3/K20). 3) We classified 9 disease types with differential HM cycle expression pattern. Eleven disease conditions presented most 4 HM cycle pathway suppression. 4) Three disease conditions had all 4 HM cycle pathway suppression and only histone hypomethylation on H3R2/K4/R8/K9/K36 and H4R3. 5) Eleven HM cycle metabolic enzymes interact with 955 proteins. 6) Five paired HM cycle proteins interact with each other. We conclude that HM cycle is a key metabolic sensor system which mediates receptor-independent metabolism-associated danger signal recognition and modulates SAM/SAH-dependent methylation in disease conditions and that hypomethylation on frequently modified histone residues is a key mechanism for metabolic disorders, autoimmune disease and CVD. We propose that HM metabolism takes place in the cytosol, that nuclear methylation equilibration requires a nuclear-cytosol transfer of SAM/SAH/Hcy, and that Hcy clearance is essential for genetic protection.

Twenty Novel Disease Group-Specific and 12 New Shared Macrophage Pathways in Eight Groups of 34 Diseases Including 24 Inflammatory Organ Diseases and 10 Types of Tumors.

The mechanisms underlying pathophysiological regulation of tissue macrophage (Mφ) subsets remain poorly understood. From the expression of 207 Mφ genes comprising 31 markers for 10 subsets, 45 transcription factors (TFs), 56 immunometabolism enzymes, 23 trained immunity (innate immune memory) enzymes, and 52 other genes in microarray data, we made the following findings. (1) When 34 inflammation diseases and tumor types were grouped into eight categories, there was differential expression of the 31 Mφ markers and 45 Mφ TFs, highlighted by 12 shared and 20 group-specific disease pathways. (2) Mφ in lung, liver, spleen, and intestine (LLSI-Mφ) express higher M1 Mφ markers than lean adipose tissue Mφ (ATMφ) physiologically. (3) Pro-adipogenic TFs C/EBPα and PPARγ and proinflammatory adipokine leptin upregulate the expression of M1 Mφ markers. (4) Among 10 immune checkpoint receptors (ICRs), LLSI-Mφ and bone marrow (BM) Mφ express higher levels of CD274 (PDL-1) than ATMφ, presumably to counteract the M1 dominant status via its reverse signaling behavior. (5) Among 24 intercellular communication exosome mediators, LLSI- and BM- Mφ prefer to use RAB27A and STX3 than RAB31 and YKT6, suggesting new inflammatory exosome mediators for propagating inflammation. (6) Mφ in peritoneal tissue and LLSI-Mφ upregulate higher levels of immunometabolism enzymes than does ATMφ. (7) Mφ from peritoneum and LLSI-Mφ upregulate more trained immunity enzyme genes than does ATMφ. Our results suggest that multiple new mechanisms including the cell surface, intracellular immunometabolism, trained immunity, and TFs may be responsible for disease group-specific and shared pathways. Our findings have provided novel insights on the pathophysiological regulation of tissue Mφ, the disease group-specific and shared pathways of Mφ, and novel therapeutic targets for cancers and inflammations.

Experimental Data-Mining Analyses Reveal New Roles of Low-Intensity Ultrasound in Differentiating Cell Death Regulatome in Cancer and Non-cancer Cells via Potential Modulation of Chromatin Long-Range Interactions.

Background: The mechanisms underlying low intensity ultrasound (LIUS) mediated suppression of inflammation and tumorigenesis remain poorly determined. Methods: We used microarray datasets from NCBI GEO Dataset databases and conducted a comprehensive data mining analyses, where we studied the gene expression of 299 cell death regulators that regulate 13 different cell death types (cell death regulatome) in cells treated with LIUS. Results: We made the following findings: (1) LIUS exerts a profound effect on the expression of cell death regulatome in cancer cells and non-cancer cells. Of note, LIUS has the tendency to downregulate the gene expression of cell death regulators in non-cancer cells. Most of the cell death regulator genes downregulated by LIUS in non-cancer cells are responsible for mediating inflammatory signaling pathways; (2) LIUS activates different cell death transcription factors in cancer and non-cancer cells. Transcription factors TP-53 and SRF- were induced by LIUS exposure in cancer cells and non-cancer cells, respectively; (3) As two well-accepted mechanisms of LIUS, mild hyperthermia and oscillatory shear stress induce changes in the expression of cell death regulators, therefore, may be responsible for inducing LIUS mediated changes in gene expression patterns of cell death regulators in cells; (4) LIUS exposure may change the redox status of the cells. LIUS may induce more of antioxidant effects in non-cancer cells compared to cancer cells; and (5) The genes modulated by LIUS in cancer cells have distinct chromatin long range interaction (CLRI) patterns to that of non-cancer cells. Conclusions: Our analysis suggests novel molecular mechanisms that may be utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Increased acetylation of H3K14 in the genomic regions that encode trained immunity enzymes in lysophosphatidylcholine-activated human aortic endothelial cells - Novel qualification markers for chronic disease risk factors and conditional DAMPs.

To test our hypothesis that proatherogenic lysophosphatidylcholine (LPC) upregulates trained immunity pathways (TIPs) in human aortic endothelial cells (HAECs), we conducted an intensive analyses on our RNA-Seq data and histone 3 lysine 14 acetylation (H3K14ac)-CHIP-Seq data, both performed on HAEC treated with LPC. Our analysis revealed that: 1) LPC induces upregulation of three TIPs including glycolysis enzymes (GE), mevalonate enzymes (ME), and acetyl-CoA generating enzymes (ACE); 2) LPC induces upregulation of 29% of 31 histone acetyltransferases, three of which acetylate H3K14; 3) LPC induces H3K14 acetylation (H3K14ac) in the genomic DNA that encodes LPC-induced TIP genes (79%) in comparison to that of in LPC-induced effector genes (43%) including ICAM-1; 4) TIP pathways are significantly different from that of EC activation effectors including adhesion molecule ICAM-1; 5) reactive oxygen species generating enzyme NOX2 deficiency decreases, but antioxidant transcription factor Nrf2 deficiency increases, the expressions of a few TIP genes and EC activation effector genes; and 6) LPC induced TIP genes(81%) favor inter-chromosomal long-range interactions (CLRI, trans-chromatin interaction) while LPC induced effector genes (65%) favor intra-chromosomal CLRIs (cis-chromatin interaction). Our findings demonstrated that proatherogenic lipids upregulate TIPs in HAECs, which are a new category of qualification markers for chronic disease risk factors and conditional DAMPs and potential mechanisms for acute inflammation transition to chronic ones. These novel insights may lead to identifications of new cardiovascular risk factors in upregulating TIPs in cardiovascular cells and novel therapeutic targets for the treatment of metabolic cardiovascular diseases, inflammation, and cancers. (total words: 245).

Co-signaling receptors regulate T-cell plasticity and immune tolerance.

We took an experimental database mining analysis to determine the expression of 28 co-signaling receptors in 32 human tissues in physiological/pathological conditions. We made the following significant findings: 1) co-signaling receptors are differentially expressed in tissues; 2) heart, trachea, kidney, mammary gland and muscle express co-signaling receptors that mediate CD4+T cell functions such as priming, differentiation, effector, and memory; 3) urinary tumor, germ cell tumor, leukemia and chondrosarcoma express high levels of co-signaling receptors for T cell activation; 4) expression of inflammasome components are correlated with the expression of co-signaling receptors; 5) CD40, SLAM, CD80 are differentially expressed in leukocytes from patients with trauma, bacterial infections, polarized macrophages and in activated endothelial cells; 6) forward and reverse signaling of 50% co-inhibition receptors are upregulated in endothelial cells during inflammation; and 7) STAT1 deficiency in T cells upregulates MHC class II and co-stimulation receptors. Our results have provided novel insights into co-signaling receptors as physiological regulators and potentiate identification of new therapeutic targets for the treatment of sterile inflammatory disorders.

DNA Checkpoint and Repair Factors Are Nuclear Sensors for Intracellular Organelle Stresses-Inflammations and Cancers Can Have High Genomic Risks.

Under inflammatory conditions, inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) which cause DNA damage. If not appropriately repaired, DNA damage leads to gene mutations and genomic instability. DNA damage checkpoint factors (DDCF) and DNA damage repair factors (DDRF) play a vital role in maintaining genomic integrity. However, how DDCFs and DDRFs are modulated under physiological and pathological conditions are not fully known. We took an experimental database analysis to determine the expression of 26 DNA DDCFs and 42 DNA DDRFs in 21 human and 20 mouse tissues in physiological/pathological conditions. We made the following significant findings: (1) Few DDCFs and DDRFs are ubiquitously expressed in tissues while many are differentially regulated.; (2) the expression of DDCFs and DDRFs are modulated not only in cancers but also in sterile inflammatory disorders and metabolic diseases; (3) tissue methylation status, pro-inflammatory cytokines, hypoxia regulating factors and tissue angiogenic potential can determine the expression of DDCFs and DDRFs; (4) intracellular organelles can transmit the stress signals to the nucleus, which may modulate the cell death by regulating the DDCF and DDRF expression. Our results shows that sterile inflammatory disorders and cancers increase genomic instability, therefore can be classified as pathologies with a high genomic risk. We also propose a new concept that as parts of cellular sensor cross-talking network, DNA checkpoint and repair factors serve as nuclear sensors for intracellular organelle stresses. Further, this work would lead to identification of novel therapeutic targets and new biomarkers for diagnosis and prognosis of metabolic diseases, inflammation, tissue damage and cancers.

Anatomic Limitations of the Transmetatarsal Amputation With Consideration of the Deep Plantar Perforating Branch of the Dorsalis Pedis Artery.

The transmetatarsal amputation is considered a durable procedure with respect to limb salvage when managing the consequences of diabetic foot disease. The success of the procedure is, in part, determined by the preoperative appreciation of arterial and functional status. The objectives of the present investigation were to determine the length of the remaining first metatarsal required during transmetatarsal amputation to preserve the anastomotic connection of the deep plantar perforating artery and subsequent "vascular arch" of the foot and the insertion of the tibialis anterior tendon. The primary outcome measure of our investigation was a measurement of the distance between the first metatarsal-medial cuneiform articulation and the distal extent of the deep plantar perforating artery in 85 embalmed lower limbs. As a secondary outcome measure, the insertion of the tibialis anterior tendon was evaluated relative to the deep plantar perforating artery. The most distal extent of the deep plantar perforating artery was observed at a mean ± standard deviation of 15.62 ± 3.74 (range 6.0 to 28.28) mm from the first metatarsal-medial cuneiform articulation. Most (89.41%) of the arteries were found within 20 mm of the first metatarsal-medial cuneiform articulation. The insertion of the tibialis anterior tendon was found to be proximal to the deep plantar perforating artery in all specimens (100.0%). In conclusion, 2.0 cm of remnant first metatarsal might represent an anatomic definition of how "short" a transmetatarsal amputation can safely be performed in most patients when considering the vascular and biomechanical anatomy.