Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that regulates gene expression, and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its roles in endothelial cells (ECs), angiogenesis, and vascular function. Using inducible EC-specific TDP-43-KO mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity, and blood vessel stability. Postnatal EC-specific deletion of TDP-43 led to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the CNS with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted the fibronectin matrix around sprouting vessels and reduced ß-catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.

Deletion of endothelial α-parvin inhibits tumour angiogenesis, reduces tumour growth and induces tumour cell apoptosis.

Alpha-parvin (α-pv), an adaptor protein that mediates integrin-dependent cell-matrix interactions, is essential for endothelial cells migration and proliferation and is a key player in physiological angiogenesis. The role of α-pv in pathological angiogenesis is unknown. Here we demonstrate that endothelial α-pv is required for tumour angiogenesis. Using an inducible knockout approach in which the α-pv gene (Parva) was inactivated specifically in endothelial cells of brain tumour-bearing mice, we show that loss of endothelial α-pv results in reduced vessel density and decreased vascular complexity of the pathological neo-vasculature without affecting the structure of the brain vasculature around tumour. Reduced tumour vascularisation is associated with a significant increase in tumour cell apoptosis and a reduction in tumour volume. Together, our data show for the first time that endothelial α-pv is required for tumour vascularisation and tumour progression in vivo.

The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain.

Nerve injury produces neuropathic pain through the binding of α2δ1 proteins to glutamate N-methyl-D-aspartate receptors (NMDARs). Notably, mice with a targeted deletion of the sigma 1 receptor (σ1R) gene do not develop neuropathy, whereas mice lacking the histidine triad nucleotide-binding protein 1 (Hint1) gene exhibit exacerbated allodynia. σ1R antagonists more effectively diminish neuropathic pain of spinal origin when administered by intracerebroventricular injection than systemically. Thus, in mice subjected to unilateral sciatic nerve chronic constriction injury (CCI), we studied the participation of σ1Rs and HINT1 proteins in the formation of α2δ1-NMDAR complexes within the supraspinal periaqueductal gray (PAG). We found that δ1 peptides required σ1Rs in order to interact with the NMDAR NR1 variant that contains the cytosolic C1 segment. σ1R antagonists or low calcium levels provoke the dissociation of σ1R-NR1 C1 dimers, while they barely affect the integrity of δ1-σ1R-NR1 C1 trimers. However, HINT1 does remove δ1 peptides from the trimer, thereby facilitating the subsequent dissociation of σ1Rs from NMDARs. In σ1R-/- mice, CCI does not promote the formation of NMDAR-α2δ1 complexes and allodynia does not develop. The levels of α2δ1-σ1R-NMDAR complexes increase in HINT1-/- mice and after inducing CCI, degradation of α2δ1 proteins is observed. Notably, σ1R antagonists but not gabapentinoids alleviate neuropathic pain in these mice. During severe neuropathy, the metabolism of α2δ1 proteins may account for the failure of many patients to respond to gabapentinoids. Therefore, σ1Rs promote and HINT1 proteins hinder the formation α2δ1-NMDAR complexes in the PAG, and hence, the appearance of mechanical allodynia depends on the interplay between these proteins.

Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels.

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R-NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R-binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R-NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R-BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.

Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor.

Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R-/- mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.

Mediterranean tomato-based sofrito protects against vascular alterations in obese Zucker rats by preserving NO bioavailability.

SCOPE: Sofrito, a key component of the Mediterranean diet, provides nutritional interest due to its high content in bioactive compounds from tomato and olive oil, and especially to the lipid matrix in which these compounds are found. In this study, the potential beneficial effects of dietary intake of sofrito on obesity-related vascular alterations were explored in obese Zucker rats. METHODS AND RESULTS: Obese and lean rats were fed a control diet supplemented or not with 2% w/w sofrito for 8 weeks. Vascular function was evaluated in aorta in organ baths. Dihydroethidium staining and immunofluorescence was used to determine aortic superoxide and peroxynitrite production, respectively. Despite food and caloric intake was higher in sofrito-fed obese rats, no differences were appreciated on body weight compared to control rats. Sofrito attenuated phenylephrine-induced vasoconstriction. This effect was associated with preservation of nitric oxide on vasoconstriction and normalization of serum nitric oxide metabolites, vascular inducible nitric oxide synthase and vascular superoxide and peroxynitrite levels. CONCLUSION: This is the first evidence of tomato-based sofrito protection against vascular alterations that could precede major cardiometabolic complications in obesity. These results contribute to explain the therapeutic properties of the Mediterranean diet in obesity-related disorders. Therefore, sofrito is an attractive dietary approach against vascular alterations in obesity.

Sex differences in angiotensin II responses contribute to a differential regulation of cox-mediated vascular dysfunction during aging.

Aging is a cardiovascular risk factor partially related to activation of the Renin-Angiotensin System (RAS). RAS activation is also influenced by sex. In this regard, our study aims to determine whether sex-associated differences in RAS contribute to a differential regulation of vascular aging and associated dysfunction. Male and female outbreed CD-1 mice were studied at 3 and 12months of age (M). Contribution of RAS was determined by treating mice from 3M to 12M with the AngII type 1 receptor blocker losartan (0.6g/L in the drinking water). At 12M, contractions to AngII were higher in males compared to females (P<0.05). This effect was paralleled by a decrease in AngII type 2 receptors in 12M males. Aging also diminished ACh relaxation in males, but did not modify female responses. Treatment of aortas with indomethacin (10µM) restored the impaired endothelium-dependent relaxation in 12M males, suggesting an increase of cyclooxygenase (COX)-derived vasoconstrictors in aged males. Chronic treatment of mice with losartan also improved endothelium-dependent relaxation. Besides, losartan significantly decreased COX-2 expression and activity in 12M male, with a minor effect in aged females. Aging increases AngII contraction and induces endothelial dysfunction differently in males and females. In aged males, RAS contributed to increased COX-2 expression and activity, which in turn may lead to vascular dysfunction.

NADPH oxidase 4 attenuates cerebral artery changes during the progression of Marfan syndrome.

Marfan syndrome (MFS) is a connective tissue disorder that is often associated with the fibrillin-1 (Fbn1) gene mutation and characterized by cardiovascular alterations, predominantly ascending aortic aneurysms. Although neurovascular complications are uncommon in MFS, the improvement in Marfan patients' life expectancy is revealing other secondary alterations, potentially including neurovascular disorders. However, little is known about small-vessel pathophysiology in MFS. MFS is associated with hyperactivated transforming growth factor (TGF)-ß signaling, which among numerous other downstream effectors, induces the NADPH oxidase 4 (Nox4) isoform of NADPH oxidase, a strong enzymatic source of H2O2 We hypothesized that MFS induces middle cerebral artery (MCA) alterations and that Nox4 contributes to them. MCA properties from 3-, 6-, or 9-mo-old Marfan (Fbn1(C1039G/+)) mice were compared with those from age/sex-matched wild-type littermates. At 6 mo, Marfan compared with wild-type mice developed higher MCA wall/lumen (wild-type: 0.081 ± 0.004; Marfan: 0.093 ± 0.002; 60 mmHg; P < 0.05), coupled with increased reactive oxygen species production, TGF-ß, and Nox4 expression. However, wall stiffness and myogenic autoregulation did not change. To investigate the influence of Nox4 on cerebrovascular properties, we generated Marfan mice with Nox4 deficiency (Nox4(-/-)). Strikingly, Nox4 deletion in Marfan mice aggravated MCA wall thickening (cross-sectional area; Marfan: 6,660 ± 363 µm(2); Marfan Nox4(-/-): 8,795 ± 824 µm(2); 60 mmHg; P < 0.05), accompanied by decreased TGF-ß expression and increased collagen deposition and Nox1 expression. These findings provide the first evidence that Nox4 mitigates cerebral artery structural changes in a murine model of MFS.

Middle cerebral artery remodeling following transient brain ischemia is linked to early postischemic hyperemia: a target of uric acid treatment.

Ischemia impairs blood supply to the brain, and reperfusion is important to restore cerebral blood flow (CBF) and rescue neurons from cell death. However, reperfusion can induce CBF values exceeding the basal values before ischemia. This hyperemic effect has been associated with a worse ischemic brain damage, albeit the mechanisms that contribute to infarct expansion are not clear. In this study, we investigated the influence of early postischemic hyperemia on brain damage and middle cerebral artery (MCA) properties and the effect of treatment with the endogenous antioxidant uric acid (UA). The MCA was occluded for 90 min followed by 24 h reperfusion in adult male Sprague-Dawley rats. Cortical CBF increases at reperfusion beyond 20% of basal values were taken as indicative of hyperemia. UA (16 mg/kg) or vehicle (Locke's buffer) was administered intravenously 135 min after MCA occlusion. Hyperemic compared with nonhyperemic rats showed MCA wall thickening (sham: 22.4 ± 0.8 µm; nonhyperemic: 23.1 ± 1.2 µm; hyperemic: 27.8 ± 0.9 at 60 mmHg; P < 0.001, hyperemic vs. sham) involving adventitial cell proliferation, increased oxidative stress, and interleukin-18, and more severe brain damage. Thus MCA remodeling after ischemia-reperfusion takes place under vascular oxidative and inflammatory stress conditions linked to hyperemia. UA administration attenuated MCA wall thickening, induced passive lumen expansion, and reduced brain damage in hyperemic rats, although it did not increase brain UA concentration. We conclude that hyperemia at reperfusion following brain ischemia induces vascular damage that can be attenuated by administration of the endogenous antioxidant UA.

Western diet consumption promotes vascular remodeling in non-senescent mice consistent with accelerated senescence, but does not modify vascular morphology in senescent ones.

Senescence accelerated mice (SAM) are susceptible to developing vascular dysfunction and remodeling. Food intake and type of diet have also been identified as determining factors in vascular remodeling. However, the interplay between senescence and diet in vascular remodeling is largely unknown. We aimed to analyze structure of large (aorta) and small (mesenteric; MA) arteries from seven-month-old SAM prone (SAMP8) and resistant (SAMR1) mice that received a Western-type high-fat diet (WD; 8weeks). Aortic structure was assessed by morphometric analysis of hematoxylin and eosin-stained cross sections, and collagen content by qRT-PCR, immunofluorescence and picrosirius red. In MAs, structural and mechanical properties were measured by pressure myography; elastin and collagen content by qRT-PCR and immunofluorescence; nuclei distribution by confocal microscopy; and apoptosis by qRT-PCR and TUNEL assay. In aorta, wall thickness (WT), but not cross-sectional area (CSA), was increased by senescence, and WD only increased WT in SAMR1. WD intake, but not senescence, was associated with increased collagen deposition. In MAs, senescence diminished WT and CSA, without altering collagen and elastin deposition, reduced the number of MA wall cells, and increased pro apoptotic activation. WD consumption promoted in SAMR1 the same remodeling observed with senescence, while in SAMP8 the senescence-associated changes remained unaffected. The mechanisms involved in WD-induced MA remodeling in SAMR1 mimicked those observed in senescence per se. Our study reveals qualitatively different remodeling in aortas and MAs from senescent mice. Consumption of a WD induced remodeling of the SAMR1 vasculature similar to that induced by senescence, while it did not promote any further alteration in the latter. Therefore, we propose that increased consumption of fat-enriched diets could promote accelerated senescence of the non-senescent vasculature, although it does not exacerbate vascular remodeling during senescence.

Western-type diet induces senescence, modifies vascular function in non-senescence mice and triggers adaptive mechanisms in senescent ones.

The effects of high-fat diet ingestion on senescence-induced modulation of contractile responses to phenylephrine (Phe) were determined in aortas of senescence-accelerated (SAMP8) and non-senescent (SAMR1) mice fed (8weeks) a Western-type high-fat diet (WD). Increased levels of senescence-associated ß-galactosidase staining were found in aortas of SAMP8 and SAMR1 with WD. In SAMR1, WD did not modify Phe contraction in spite of inducing major changes in the mechanisms of regulation of contractile responses. Although WD increased NAD(P)H-oxidase-derived O2(-) and augmented peroxynitrite formation, we found an increase of inducible NOS (iNOS)-derived NO production which may contribute to maintain Phe contraction in SAMR1 WD. On SAMP8, WD significantly decreased Phe-induced contractions when compared with SAMP8 under normal chow. This response was not dependent on changes of NOS expression, but rather as consequence of increased antioxidant capacity by superoxide dismutase (SOD1). A similar constrictor influence from cyclooxygenase (COX) pathway on Phe responses was found in SAMR1 and SAMP8 ND. However, WD removed that influence on SAMR1, and produced a switch in the balance from a vasoconstrictor to a vasodilator component in SAMP8. These results were associated to the increased COX-2 expression, suggesting that a COX-2-derived vasodilator prostaglandin may contribute to the vascular adaptations after WD intake. Taken together, our data suggest that WD plays a detrimental role in the vasculature of non-senescent mice by increasing pro-inflammatory (iNOS) and pro-oxidative signaling pathways and may contribute to increase vascular senescence. In senescent vessels, however, WD triggers different intrinsic compensatory alterations which include increase of antioxidant activity by SOD1 and vasodilator prostaglandin production via COX-2.

Western-style diet modulates contractile responses to phenylephrine differently in mesenteric arteries from senescence-accelerated prone (SAMP8) and resistant (SAMR1) mice.

The influence of two known cardiovascular risk factors, aging and consumption of a high-fat diet, on vascular mesenteric artery reactivity was examined in a mouse model of accelerated senescence (SAM). Five-month-old SAM prone (SAMP8) and resistant (SAMR1) female mice were fed a Western-type high-fat diet (WD; 8 weeks). Mesenteric arteries were dissected, and vascular reactivity, protein and messenger RNA expression, superoxide anion (O 2 (·-) ) and hydrogen peroxide formation were evaluated by wire myography, immunofluorescence, RT-qPCR, ethidium fluorescence and ferric-xylenol orange, respectively. Contraction to KCl and relaxation to acetylcholine remained unchanged irrespective of senescence and diet. Although similar contractions to phenylephrine were observed in SAMR1 and SAMP8, accelerated senescence was associated with decreased eNOS and nNOS and increased O 2 (·-) synthesis. Senescence-related alterations were compensated, at least partly, by the contribution of NO derived from iNOS and the enhanced endogenous antioxidant capacity of superoxide dismutase 1 to maintain vasoconstriction. Administration of a WD induced qualitatively different alterations in phenylephrine contractions of mesenteric arteries from SAMR1 and SAMP8. SAMR1 showed increased contractions partly as a result of decreased NO availability generated by decreased eNOS and nNOS and enhanced O 2 (·-) formation. In contrast, WD feeding in SAMP8 resulted in reduced contractions due to, at least in part, the increased functional participation of iNOS-derived NO. In conclusion, senescence-dependent intrinsic alterations during early stages of vascular senescence may promote vascular adaptation and predispose to further changes in response to high-fat intake, which may lead to the progression of aging-related cardiovascular disease, whereas young subjects lack the capacity for this adaptation.