Search

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8–12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31+/BrdU+ cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO+ cells), NETs (MPO+/CitH3+/DAPI+), and macrophages (CD68+ cells) in SV-129 mice, whereas macrophage polarization (MRC1- vs. MRC1+) and total leukocyte infiltration (CD45+ cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31+/CD45−/BrdU+). This was accompanied by a reduction of total leukocyte count (CD45+), neutrophils (MPO+), neutrophil extracellular traps (NETs) (MPO+CitH3+), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68+/MRC1−) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68+/MRC1+) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&amp, E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31+/BrdU+). Moreover, our results showed that the total number of leukocytes (CD45+) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO+), neutrophil extracellular trap formation (MPO+/CitH3+), and macrophages (CD68+) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68+/MRC1+). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis.

The giant 358-kDa protein Ran binding protein 2 (RanBP2/Nup358) is localized at the cytoplasmic side of the nuclear pore complex and likely constitutes the Ran-GTP binding site at the cytoplasmic face of the complex. RanBP2/Nup358 furthermore acts as a chaperone for red/green opsin molecules. Here, we report on the physical mapping of human RanBP2 between markers D2S340 and D2S1893. A duplication of the 5'-end sequence of RanBP2 occurs within 3 Mb distal to RanBP2. Detailed sequence analysis resulted in primers specific for this distal duplication. Polymerase chain reaction-based screening of cDNA libraries indicates that this transcript, called RanBP2alpha (HGMW-approved symbol RANBP2L1), is expressed in several tissues. Screening of a fetal brain cDNA library yielded a 4057-bp partial cDNA clone for RanBP2alpha. Its 5'-end is almost identical to RanBP2, whereas its 3'-part is distinct from RanBP2. Northern blot analysis using a probe of the 3'-untranslated sequence of RanBP2alpha detected in several tissues an 8-kb transcript representing the full length of the transcript. In pancreas and placenta, an additional transcript of 14 kb was detected. PAC clones containing the bona fide RanBP2 sequences were localized to 2q11-q12 by FISH analysis, and a region of high similarity was detected on 2p11-p12. In summary, we have identified a RanBP2 gene cluster on 2q11-q12 together with a novel gene termed RanBP2alpha, with high sequence similarity to RanBP2.

A gene for the autosomal recessive kidney disorder juvenile nephronophthisis (NPH) is located on chromosome 2q between markers D2S1893 and D2S1888. Recently, the presence of large homozygous deletions was described in the majority of NPH patients. We constructed an integrated YAC/PAC contig of 54 markers and 30 PAC clones that encompasses this deletion and the flanking inverted duplication. Thirty-six novel sequence-tagged site markers were generated for this region of 2–3 Mb, 22 of which represent PAC ends. Ten of 18 multiplex NPH families show a homozygous deletion for 8 consecutive markers. BlastN database search and expressed sequence tag (EST) mapping led to the localization of 18 EST clones to the integrated contig, representing 11 putative transcribed sequences. Seven EST clones were localized to the NPHP1 region between D2S1893 and D2S1888. Two EST clones, zc07a11 from a human parathyroid tumor library and yy63e10 from a multiple sclerosis lesion library, are located in the deletion region. PCR amplification experiments indicate that zc07a11 represents a chimeric cDNA. Through FISH analysis the NPHP1 deletion region was localized to 2q12–q13. In summary, our study provides a high-resolution physical map of the NPHP1 region with 7 precisely localized expressed sequences, 2 of which have recently been shown to be part of a gene for NPH. These data will alleviate the identification of further genes of a homozygous gene deletion syndrome in patients with NPH and oculomotor apraxia and will be instrumental in the characterization of the molecular mechanism leading to the large homozygous deletion in this region. The data furthermore provide an important step toward the construction of a sequence-ready PAC contig of this region.

Genes of the interleukin-1 (IL-1) gene cluster localized on chromosome 2q13 are implicated in many physiological and pathophysiological processes. We present here a high-resolution physical map of this region between markers D2S2008 and D2S43/PAX8. An integrated YAC/PAC contig and a partial transcriptional map were constructed by STS-content mapping using the CEPH YAC library and three PAC libraries. A total of 3 YACs, 34 PACs, and 56 STSs were integrated: 33 newly generated probes to PAC end sequences, 9 polymorphic and 4 nonpolymorphic markers, 5 known genes, 4 expressed sequence tags, and 1 pseudogene. Within the map, a complete PAC contig of >1 Mb encompasses the IL-1 gene cluster and PAX8, a paired-box-containing gene. This allowed us to define the transcriptional orientation of GLVR1, IL1B, and IL1RN and to show that PAX8 is localized outside the IL-1 gene cluster. FISH analysis localized PAC clones containing the IL-1 gene cluster to 2q12–q13. The data provide the basis for further characterization of the IL-1 gene cluster and for the construction of a sequence-ready PAC contig of this region.