Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

Electrostatically assembled wound dressings deliver pro-angiogenic anti-miRs preferentially to endothelial cells.

Chronic non-healing wounds occur frequently in individuals affected by diabetes, yet standard-of-care treatment leaves many patients inadequately treated or with recurring wounds. MicroRNA (miR) expression is dysregulated in diabetic wounds and drives an anti-angiogenic phenotype, but miRs can be inhibited with short, chemically-modified RNA oligonucleotides (anti-miRs). Clinical translation of anti-miRs is hindered by delivery challenges such as rapid clearance and uptake by off-target cells, requiring repeated injections, excessively large doses, and bolus dosing mismatched to the dynamics of the wound healing process. To address these limitations, we engineered electrostatically assembled wound dressings that locally release anti-miR-92a, as miR-92a is implicated in angiogenesis and wound repair. In vitro, anti-miR-92a released from these dressings was taken up by cells and inhibited its target. An in vivo cellular biodistribution study in murine diabetic wounds revealed that endothelial cells, which play a critical role in angiogenesis, exhibit higher uptake of anti-miR eluted from coated dressings than other cell types involved in the wound healing process. In a proof-of-concept efficacy study in the same wound model, anti-miR targeting anti-angiogenic miR-92a de-repressed target genes, increased gross wound closure, and induced a sex-dependent increase in vascularization. Overall, this proof-of-concept study demonstrates a facile, translational materials approach for modulating gene expression in ulcer endothelial cells to promote angiogenesis and wound healing. Furthermore, we highlight the importance of probing cellular interactions between the drug delivery system and the target cells to drive therapeutic efficacy.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

miR-181b regulates vascular endothelial aging by modulating an MAP3K3 signaling pathway.

Endothelial cell (EC) aging plays a vital role in the pathogenesis of cardiovascular disease (CVD). MicroRNAs have emerged as crucial regulators of target gene expression by inhibiting mRNA translation and/or promoting mRNA degradation. We identify an aging-related and oxidative stress-responsive microRNA, miR-181b, that inhibits endothelial cell apoptosis and senescence. In gain- or loss-of-function studies, miR-181b regulated the expression of key apoptosis markers (Bcl2, Bax, cleaved-Caspase3) and senescence markers (p16, p21, γH2AX) and the ratio of apoptotic cells (TUNEL-positive) and senescent cells (SA-ßgal-positive) in H2 O2 -induced ECs. Mechanistically, miR-181b targets MAP3K3 and modulates a MAP3K3/MKK/MAPK signaling pathway. MAP3K3 knockdown recapitulated the phenotype of miR-181b overexpression and miR-181b was dependent on MAP3K3 for regulating EC apoptosis and senescence. In vivo, miR-181b expression showed a negative correlation with increasing age in the mouse aorta. Endothelial-specific deficiency of miR-181a2b2 increased the target MAP3K3, markers of vascular senescence (p16, p21), and DNA double-strand breaks (γH2AX) in the aorta of aged mice. Collectively, this study unveils an important role of miR-181b in regulating vascular endothelial aging via an MAP3K3-MAPK signaling pathway, providing new potential therapeutic targets for antiaging therapy in CVD.

A miRNA cassette reprograms smooth muscle cells into endothelial cells.

Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4-miRNA cassette consisting of miR-143-3p and miR-145-5p inhibitors and miR-146a-5p and miR-181b-5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4-miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.

Deficiency of lncRNA SNHG12 impairs ischemic limb neovascularization by altering an endothelial cell cycle pathway.

SNHG12, a long noncoding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease has not been elucidated. Hind-limb ischemia studies using femoral artery ligation (FAL) in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain- and loss-of-function studies demonstrated that SNHG12 regulated angiogenesis - SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including IGF-2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3). RNA-Seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IMP3. Knockdown of SNHG12 in mice undergoing FAL using injected gapmeRs) decreased angiogenesis, an effect that was more pronounced in a model of insulin-resistant db/db mice. RNA-Seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12 knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.

Endothelial TLR2 promotes proangiogenic immune cell recruitment and tumor angiogenesis.

Toll-like receptor 2 (TLR2) is implicated in various pathologies, mainly in terms of its function within innate immune cells. However, TLR2 is also present in endothelial cells. Here, we explored the physiological and pathophysiological roles of endothelial TLR2 signaling. We found that TLR2 was highly abundant in the endothelium within various tissues using TLR2-IRES-EGFP reporter mice and was required for proinflammatory endothelial cell function. Endothelial cells lacking TLR2 exhibited reduced proinflammatory potential at the protein, cell, and tissue levels. Loss of endothelial TLR2 blunted the inflammatory response to both exogenous and endogenous danger signals in endothelial cells in culture and in vivo. Endothelial TLR2 promoted tumor growth, angiogenesis, and protumorigenic immune cell recruitment in a mouse model of prostate cancer. Furthermore, the cell surface localization of P-selectin and the subsequent production of other critical cell adhesion molecules (such as E-selectin, ICAM-1 and VCAM-1) that recruit immune cells required endothelial TLR2. Our findings demonstrate that endothelial cells actively contribute to innate immune pathways and propose that endothelial TLR2 has a pathological role in proinflammatory conditions.

Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Collagen Fiber Orientation Regulates 3D Vascular Network Formation and Alignment.

Alignment of collagen type I fibers is a hallmark of both physiological and pathological tissue remodeling. However, the effects of collagen fiber orientation on endothelial cell behavior and vascular network formation are poorly understood because of a lack of model systems that allow studying these potential functional connections. By casting collagen type I into prestrained (0, 10, 25, 50% strain), poly(dimethylsiloxane) (PDMS)-based microwells and releasing the mold strain following polymerization, we have created collagen gels with varying fiber alignment as confirmed by structural analysis. Endothelial cells embedded within the different gels responded to increased collagen fiber orientation by assembling into 3D vascular networks that consisted of thicker, more aligned branches and featured elevated collagen IV deposition and lumen formation relative to control conditions. These substrate-dependent changes in microvascular network formation were associated with altered cell division and migration patterns and related to enhanced mechanosignaling. Our studies indicate that collagen fiber alignment can directly regulate vascular network formation and that culture models with aligned collagen may be used to investigate the underlying mechanisms ultimately advancing our understanding of tissue development, homeostasis, and disease.

Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation.

UNLABELLED: Neovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches. STATEMENT OF SIGNIFICANCE: Neovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Electrospinning collagen and hyaluronic acid nanofiber meshes.

Collagen and hyaluronic acid (HA) are main components of the extracellular matrix and have been utilized in electrospinning; a technique that creates nanosized fibers for tissue scaffolds. A collagen/HA polymer solution was electrospun into a scaffold material for osteoporosis patients who have reduced bone strength. To synthesize nanofibers, a high voltage was applied to the polymer solution to draw out nanofibers that were collected on a ground plate as a uniform mesh. The meshes were then crosslinked to render them insoluble and conjugated with gold nanoparticles to promote biocompatibility. Characterization of the mesh was performed using scanning electron microscope, electron dispersive spectroscopy and fourier transform infrared spectroscopy. A WST-1 assay determined the potential biocompatibility. The results show that collagen/HA scaffolds were developed that were insoluble in aqueous solutions and promoted cellular attachment that could be used as a tissue engineered scaffold to promote cell growth.