Local Secretion of Urocortin 1 Promotes Microvascular Permeability during Lipopolysaccharide-Induced Inflammation

Urocortin 1 (Ucn1) is a neuropeptide that regulates vascular tone and is implicated in both the vascular and immune cell-mediated responses to inflammation. The role of Ucn1 in regulating microvascular permeability has not been determined. We hypothesized that local Ucn1 release promotes microvascular permeability and that this effect augments the local gastrointestinal vascular response to lipopolysaccharide (LPS)-induced systemic inflammation. We measured hydraulic (Lp) and macromolecule permeability in mesenteric venules. We show that a continuous infusion of 10−7m Ucn1 in a postcapillary venule increased Lp 2-fold over baseline, as did LPS-induced inflammation. However, simultaneous infusion of Ucn1 and LPS markedly increased Lp by 7-fold. After local knockdown of Ucn1 using RNA interference, infusion of Ucn1 with LPS resulted in return to 2-fold increase, confirming that Ucn1 synergistically augments hydraulic permeability during inflammation. LPS and Ucn1 treatment also resulted in increased numbers of interstitial microspheres, which colocalized with CD31+ immune cells. Ucn1 activity is mediated through two receptor subtypes, CRH-R1 and CRH-R2. CRH-R1 receptor blockade exacerbated, whereas CRH-R2 receptor blockade decreased the LPS-induced increase in Lp. Finally, treatment with the c-JUN N-terminal kinase (JNK) antagonist SP600125 during infusion of LPS, but not Ucn1, decreased Lp. These findings suggest that Ucn1 increases microvascular permeability and acts synergistically with LPS to increase fluid and macromolecule losses during inflammation. Knockdown of endogenous Ucn1 during inflammation attenuates synergistic increases in Lp. Ucn1’s effect on Lp is partially mediated by the CRH-R2 receptor and acts independently of the c-JUN N-terminal kinase signal transduction pathway.

Reperfusion-induced changes in capillary perfusion and filtration: effects of hypercholesterolemia

Fluid filtration rate ( J v/ S) and red blood cell velocity ( V RBC) in individual mesenteric capillaries of normocholesterolemic (NC) and hypercholesterolemic (HC) rats were measured before and after ischemia and reperfusion (I/R). In NC rats, a correlation was found between baseline J v/ Sand the percent of the feeding arteriole length that was paired (<15 μm) with a postcapillary venule (A-V pairing), but not in the HC group. Additionally, in NC rats only, a correlation was found between baseline V RBC and A-V pairing. In capillaries in which A-V pairing was substantial (>20%), V RBCdropped after reperfusion in the HC group (54% of baseline; P < 0.05), but not in the NC group (79%). The decrease in V RBC in HC rats could be attenuated by a P-selectin antibody (PB1.3). PB1.3 was also able to attenuate the increase in I/R-induced capillary J v/ Sin HC rats (median increase = 1.26-fold vs. 1.53-fold without PB1.3). These data suggest a role for A-V pairing in capillary perfusion in NC rats and a potential role for P-selectin in I/R-induced microvascular dysfunction in HC rats.

Nifedipine increases microvascular permeability via a direct local effect on postcapillary venules

Calcium-channel antagonist drugs are prescribed widely for angina and hypertension. A limiting side effect is edema, which can make heart failure worse. We show that nifedipine, a dihydropyridine-type calcium-channel antagonist, can increase vascular permeability in rat skeletal muscle and skin when injected locally. In nifedipine-injected cremaster muscle, the copper content, used to quantify Monastral blue dye accumulation, was 15.0 ± 2.4 μg/g compared with 5.3 ± 0.7 μg/g in control preparations ( P < 0.05). The injection of nifedipine in rat skin in vivo increased local plasma leakage in injected sites from 5.5 ± 1.1 μl in control sites to 9.9 ± 2.5, 17.0 ± 2.4, 24.3 ± 5.9, and 23.3 ± 5.4 μl in sites injected with 10−10, 10−9, 10−8, or 10−7.2 mol/site, respectively ( P < 0.05 in each case compared with control). Vascular labeling techniques using light microscopy, electron microscopy, and microanalysis show that the microvascular site of leakage is not from capillaries but from postcapillary venules of 12–36 μm in diameter, the same site that controls the edema response in inflammation. Nifedipine can act within the microcirculation to increase the permeability of the postcapillary venule.

Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium

The Duffy gene has been shown not to be split by introns, even in its 5′ untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5′-rapid amplification of cDNA ends (5′-RACE). While every positive clone of 5′- RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide - 279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in- frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.

Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium

The Duffy gene has been shown not to be split by introns, even in its 5′ untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5′-rapid amplification of cDNA ends (5′-RACE). While every positive clone of 5′- RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide - 279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in- frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.