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Improved approaches to expanding the pool of donor lungs suitable for transplantation are critically needed for the growing population with end-stage lung disease. Cross-circulation (XC) of whole blood between swine and explanted human lungs has previously been reported to enable the extracorporeal recovery of donor lungs that declined for transplantation due to acute, reversible injuries. However, immunologic interactions of this xenogeneic platform have not been characterized, thus limiting potential translational applications. Using flow cytometry and immunohistochemistry, we demonstrate that porcine immune cell and immunoglobulin infiltration occurs in this xenogeneic XC system, in the context of calcineurin-based immunosuppression and complement depletion. Despite this, xenogeneic XC supported the viability, tissue integrity, and physiologic improvement of human donor lungs over 24 hours of xeno-support. These findings provide targets for future immunomodulatory strategies to minimize immunologic interactions on this organ support biotechnology.

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe

Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.

Cell‐free hemoglobin (CFH) is associated with severe lung injury in human patients and is sufficient to induce airspace inflammation and alveolar–capillary barrier dysfunction in an experimental model of acute lung injury. The mechanisms through which this occurs are unknown. One key pathway which regulates inflammation during acute lung injury is the NLRP3 inflammasome. Because CFH can act as a damage‐associated molecular pattern, we hypothesized that CFH may activate the NLRP3 inflammasome during acute lung injury. Primary mouse alveolar macrophages and cultured murine macrophages exposed to CFH (0–1 mg/ml) for 24 hr demonstrated robust upregulation of the NLRP3 inflammasome components NLRP3, caspase‐1, and caspase‐11. Maximal induction of the NLRP3 inflammasome by CFH required TLR4. Compared to wild‐type controls, mice lacking NLRP3 developed less airspace inflammation (2.7 × 105 cells/ml in bronchoalveolar lavage fluid versus. 1.1 × 105/ml, p = .006) after exposure to intratracheal CFH. Together, these data demonstrate that CFH can stimulate the NLRP3 inflammasome in macrophages and that this pathway may be important in the pathogenesis of CFH‐induced acute lung injury., Cell‐free hemoglobin is associated with severe lung injury in human patients and is sufficient to induce airspace inflammation and alveolar–capillary barrier dysfunction in an experimental model of acute lung injury. Here, we investigate the role of the NLRP3 inflammasome in lung injury caused by cell‐free hemoglobin using in vitro and in vivo model systems. Our results show that cell‐free hemoglobin increases NLRP3 expression in alveolar macrophages and that mice lacking NLRP3 have reduced inflammation in response to cell‐free hemoglobin, suggesting that NLRP3 has an important role in acute lung injury caused by cell‐free hemoglobin.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness and commonly occurs in sepsis. Although numerous clinical and environmental risk factors exist, not all patients with risk factors develop ARDS, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels predict worse outcomes. Excess CFH is rapidly scavenged by haptoglobin (Hp). A common HP genetic variant, HP2, is unique to humans and is common in many populations worldwide. HP2 haptoglobin has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared with the alternative HP1. We hypothesized that HP2 increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared with wild-type mice. We then tested the clinical relevance of our findings in 496 septic critically ill adults, finding that HP2 increased ARDS susceptibility after controlling for clinical risk factors and plasma CFH. These observations identify HP2 as a potentially novel genetic ARDS risk factor during sepsis and may have important implications in the study and treatment of ARDS.

Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.

Primary graft dysfunction (PGD) is acute lung injury within 72 hours of lung transplantation. We hypothesized that cell-free hemoglobin (CFH) contributes to PGD by increasing lung microvascular permeability and tested this in patients, ex vivo human lungs, and cultured human lung microvascular endothelial cells. In a nested case control study of 40 patients with severe PGD at 72 hours and 80 matched controls without PGD, elevated preoperative CFH was independently associated with increased PGD risk (odds ratio [OR] 2.75, 95%CI, 1.23–6.16, P = 0.014). The effect of CFH on PGD was magnified by reperfusion fraction of inspired oxygen (FiO2) ≥ 0.40 (OR 3.41, P = 0.031). Isolated perfused human lungs exposed to intravascular CFH (100 mg/dl) developed increased vascular permeability as measured by lung weight (CFH 14.4% vs. control 0.65%, P = 0.047) and extravasation of Evans blue–labeled albumin dye (EBD) into the airspace (P = 0.027). CFH (1 mg/dl) also increased paracellular permeability of human pulmonary microvascular endothelial cell monolayers (hPMVECs). Hyperoxia (FiO2 = 0.95) increased human lung and hPMVEC permeability compared with normoxia (FiO2 = 0.21). Treatment with acetaminophen (15 μg/ml), a specific hemoprotein reductant, prevented CFH-dependent permeability in human lungs (P = 0.046) and hPMVECs (P = 0.037). In summary, CFH may mediate PGD through oxidative effects on microvascular permeability, which are augmented by hyperoxia and abrogated by acetaminophen.

MicroRNAs (miRNAs) have emerged as biomarkers of metabolic status, etiological factors in complex disease, and promising drug targets. Recent reports suggest that miRNAs are critical regulators of pathways underlying the pathophysiology of type 2 diabetes. In this study, we demonstrate by deep sequencing and real-time quantitative PCR that hepatic levels of Foxa2 mRNA and miR-29 are elevated in a mouse model of diet-induced insulin resistance. We also show that Foxa2 and miR-29 are significantly upregulated in the livers of Zucker diabetic fatty (fa/fa) rats and that the levels of both returned to normal upon treatment with the insulin-sensitizing agent pioglitazone. We present evidence that miR-29 expression in human hepatoma cells is controlled in part by FOXA2, which is known to play a critical role in hepatic energy homeostasis. Moreover, we demonstrate that miR-29 fine-tunes FOXA2-mediated activation of key lipid metabolism genes, including PPARGC1A, HMGCS2, and ABHD5. These results suggest that miR-29 is an important regulatory factor in normal metabolism and may represent a novel therapeutic target in type 2 diabetes and related metabolic syndromes.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

Atherosclerosis is associated with endothelial dysfunction and inflammation, and high-density lipoproteins (HDL) play critical roles in both. HDL transport a wide-variety of proteins, nucleic acids, and small molecules, which likely confer many of HDL’s alternative functions, including its anti-inflammatory and anti-atherogenic properties. Using in vitro studies, we demonstrated that macrophages export miR-223 to HDL and subsequently, HDL delivers functional miR-223 to human coronary artery endothelial cells (HCAEC). Moreover, intracellular adhesion molecule 1 (ICAM-1), a miR-223 target, was found to be down-regulated by HDL-miR-223 at a specific target site within ICAM-1’s 3’ untranslated region. These in vitro findings support the potential of a HDL-miRNA communication pathway that may contribute to atherogenesis. To validate HDL-miRNA communication in vivo, bone-marrow transplant (BMT) studies were completed with Mir223-/- and C57B/6J (WT) mice. To demonstrate that functional HDL-miR-223 is delivered to recipient cells/tissues in vivo, BM from WT mice was transplanted into irradiated Mir223-/- mice. Strikingly, we found that HDL-miR-223 communication was restored after BMT, as evidenced by significantly increased miR-223 levels on HDL and in the liver, white adipose, aortic endothelium, and skeletal muscle. Conversely, HDL-miR-223 intercellular communication was depleted by transplanting BM from Mir223-/- into WT mice, and resulted in significant loss of miR-223 on HDL and in many tissues. In both BMT studies, miR-223 changes led to altered expression of many target genes in multiple tissues. Most importantly, these studies identified a novel miR-223 target gene which we experimentally validated, vesicle transport through interaction with t-SNAREs homolog 1A (VTI1A), which was decreased and increased in the aortic endothelium from each BMT study, respectively. VTI1A has been previously characterized as a SNARE protein that contributes to cytokine secretion and membrane lipid dynamics. As such, HDL likely mediates an inflammatory cell-originating miR-223 communication pathway that suppresses inflammation and cytokine secretion in the aortic endothelium, thus antagonizing atherosclerosis.

In addition to cholesterol, HDL transports a wide-variety of cargo including vitamins, nucleic acids, and a diverse set of proteins. Outside of the reverse cholesterol transport pathway, HDL has alternative functions that contribute to its anti-atherogenicity, including anti-inflammatory, anti-oxidant, and signaling capacities. Recently, we found that HDL transports and delivers functional microRNAs to recipient cells, which likely confers HDL’s ability to suppress adhesion molecule expression in endothelial cells. As such, changes to HDL’s cargo impact many of these alternative functions, and thus its protective capacity. HDL dysfunction has been identified among patients with familial hypercholesterolemia (FH), an inherited disease due to mutations in the LDL receptor and associated with severe elevations in LDL-cholesterol (LDL-C) levels, which may necessitate LDL apheresis (LA) in management. Here we demonstrate that LA significantly alters HDL’s miRNA and protein signatures. We believe that these changes may have profound consequences on HDL’s protective capacity. Using density-gradient ultracentrifugation, we found 31 proteins to be significantly altered on HDL after LA, as determined by shotgun proteomics and multidimensional protein identification technology analysis. For example, vitamin D-binding protein (1.74-fold) was increased, while lipopolysaccharide-binding protein (-1.92-fold), platelet-activating factor acetylhydrolase (-2.06), and apolipoprotein A-V (-2.4-fold) were found to be decreased. Gene ontology and KEGG enrichment analysis demonstrated roles of these proteins in response to stress, coagulation, hemostasis, and vesicle-mediated transport. Fast-protein liquid chromatography was used to further purify HDL for miRNA profiling using TaqMan OpenArrays, and we found 8 HDL-miRNAs to be significantly altered -- 3 down (miR-302b -13.9-fold, miR-224 -1.8, miR-572 -2.4-fold) and 5 up (miR-7 9.8-fold, miR-208b 16.2-fold, miR-34a-3p 3-fold, miR-627 2.4-fold, miR-1183 4.8-fold). Both miR-302b and miR-224 decreased; these are miRNAs previously reported to suppress proliferation through targeting AKT2 and apoptosis, respectively. As such, these changes likely alter HDL’s function in FH subjects.

Chronic kidney disease (CKD) is associated with endothelial dysfunction and atherosclerosis. High-density lipoproteins (HDL) serve as a general cargo carrier for a wide-variety of proteins, nucleic acids, and small molecules which likely confer many of HDL’s alternative functions. Human coronary artery endothelial cells (HCAEC) treated with native HDL were found to have increased intracellular levels of miR-92a, a miRNA recently found to protect against endothelial dysfunction and atherosclerosis. Moreover, HDL delivery of miR-92a was inhibited using SR-BI blocking antibodies in endothelial cells. Likewise, aortic endothelial miR-92a levels were found to be significantly decreased (50%) in Apoe-/- mice compared to wild-type controls, which may be a function of the absence of HDL in these mice. Using photoactivatable-ribonucleoside-crosslinked-immunoprecipitation-high-throughput-sequencing, we found that HDL transfer multiple miR-92a isomiR variants from macrophages to HCAECs, which were found in association with Argonaute2 RNA-induced silencing complexes. Using whole-genome arrays, we found that HDL alters the expression of many genes in HCAECs, including the significant down-regulation of 18 putative miR-92a targets. Strikingly, miR-92a was found to be significantly increased 8.5-fold on HDL from CKD subjects. Interleukin-33 (IL-33) is a cytokine and nuclear transcription factor largely expressed in endothelial cells. HDL from both normal and CKD subjects significantly decreased IL-33 expression in HCAECs, as determined by real-time PCR and microarrays. In a mouse model of CKD (Apoe-/-; 5/6 nephrectomy), IL-33 expression was increased in aortic endothelial cells; however, a single HDL injection (4mg) reduced IL-33 mRNA back to sham levels at 7d post-injection. Most interestingly, HDL loaded with locked-nucleic acid miR-92a inhibitors blocked HDL’s ability to suppress CKD induced IL-33 expression in the aortic endothelium. IL-33 may represent a novel marker of endothelial dysfunction, as IL-33 mRNA levels were also found to be increased in HCAECs under disturbed flow conditions. Results from this study suggest that HDL suppression of IL-33 in CKD requires miR-92a, which is also transferred to endothelial cells by HDL.