MiRNA-149 as a Candidate for Facial Clefting and Neural Crest Cell Migration.

Nonsyndromic cleft lip with or without palate (nsCL/P) ranks among the most common human birth defects and has a multifactorial etiology. Human neural crest cells (hNCC) make a substantial contribution to the formation of facial bone and cartilage and are a key cell type in terms of nsCL/P etiology. Based on increasing evidence for the role of noncoding regulatory mechanisms in nsCL/P, we investigated the role of hNCC-expressed microRNAs (miRNA) in cleft development. First, we conducted a systematic analysis of miRNAs expressed in human-induced pluripotent stem cell-derived hNCC using Affymetrix microarrays on cell lines established from 4 unaffected donors. These analyses identified 152 candidate miRNAs. Based on the hypothesis that candidate miRNA loci harbor genetic variation associated with nsCL/P risk, the genomic locations of these candidates were cross-referenced with data from a previous genome-wide association study of nsCL/P. Associated variants were reanalyzed in independent nsCL/P study populations. Jointly, the results suggest that miR-149 is implicated in nsCL/P etiology. Second, functional follow-up included in vitro overexpression and inhibition of miR-149 in hNCC and subsequent analyses at the molecular and phenotypic level. Using 3'RNA-Seq, we identified 604 differentially expressed (DE) genes in hNCC overexpressing miR-149 compared with untreated cells. These included TLR4 and JUNB, which are established targets of miR-149, and NOG, BMP4, and PAX6, which are reported nsCL/P candidate genes. Pathway analyses revealed that DE genes were enriched in pathways including regulation of cartilage development and NCC differentiation. At the cellular level, distinct hNCC migration patterns were observed in response to miR-149 overexpression. Our data suggest that miR-149 is involved in the etiology of nsCL/P via its role in hNCC migration.

Functional imaging of the retinal microvasculature by scanning laser Doppler flowmetry.

PURPOSE: to image functionally perfused retinal vessels and to assess quantitatively the intercapillary space of the retinal microvasculature. METHOD: The base of functional imaging and the quantitative assessment of the retinal vasculature is the two-dimensional map of the retina encoded by the laser Doppler frequency shift. By Scanning Laser Doppler Flowmetry (HRF. Heidelberg Engineering) the laser Doppler frequency shift of 16.384 retinal sites (256 pixels x 64 lines, spatial resolution 10 mum) of a retinal area of 2.7 x 0.7 mm was gained. The image processing was performed by a recently described algorithm (AFFPIA). Using the data of the laser Doppler frequency shift of every retinal site, a color-coded retinal image was established showing perfused vessels and capillaries. By automatic pattern analysis of this image vessels and capillaries were identified and segmented. Based on this image the distances in [microm] of every retinal site to the next vessel or capillary were calculated ("distance to next capillary"). The functional imaging of the retinal perfusion was demonstrated in (1) normal retina, (2) retinal arterial occlusion, and (3) proliferative retinopathy. Intraobserver reliability of the quantitative assessment of the parameter "distance to next capillary" was estimated by measuring 10 eyes of 10 subjects at 5 different days by one observer. Interobserver reliability of the quantitative assessment was evaluated by analysing 10 perfusion maps by 5 different operators. In 93 eyes of 71 normal subjects (mean age 40.4 mu 15 years) the juxtapapillary retina was quantitatively evaluated. RESULTS: QUALITATIVE EVALUATION: The functional images of the retinal perfusion of eyes with normal retina, with retinal arterial occlusion, and with proliferative retinopathy corresponded well with the fluorescein angiography. Perfused vessels and capillaries became visible in a high local resolution. QUANTITITATIVE ASSESSMENT: The coefficient of reliability of the introobserver and interobserver reproducibility of the parameter 'mean distance to next capillary" was 0.74, and 0.95, respectively. The quantitative assessment of the perfusion showed that the major part of the retinal sites (>700%) had distances to the next capillary lower than 30 microm 46% of the retinal area had distances to the next capillary from 0-20 microm 26% of the retina had distances from 20-30 microm, 12% of the retina had distances from 30-40 microm 7% of the retina had distances from 40-50 microm, 4% of the retina had distances from 50-60 microm, and 4% of the retinal sites showed distances to the next capillary greater than 60 mum. The mean distance to the next capillary or vessel was calculated with 21 +/- 6.5 microm. CONCLUSION: By non-invasive Scanning Laser Doppler Flowmetry in combination with adequate software it is possible to perform a functional imaging of the retinal vasculature and to measure all index for the functional density of retinal capillaries and vessels.

Automatic full field analysis of perfusion images gained by scanning laser Doppler flowmetry.

BACKGROUND: Scanning laser Doppler flowmetry (SLDF) enables the measurement of the laser Doppler frequency shift in retinal tissue. This process allows the quantification of retinal and optic nerve head perfusion in an area of 2.7 mm x 0.7 mm within 2 seconds and with a spatial resolution of 10 microns x 10 microns. Owing to the local heterogeneity of the retinal microcirculation itself and to heart associated pulsation the capillary retinal blood flow depends on location and time. Because of technical limitations measurements of flow are only valid in retinal points with adequate brightness and focus, and away from big vessels. To include the heart beat associated pulsation and the spatial heterogeneity of retinal blood flow into the evaluation of blood flow an algorithm was developed examining automatically the whole SLDF perfusion image. AIM: To report intraobserver reliability and interobserver reliability of a new method for analysing automatically full field perfusion images. METHOD: The base of blood flow calculation by the automatic full field perfusion image analyser (AFFPIA) was 16,384 intensity time curves of all pixels of the whole perfusion image gained by the SLDF. AFFPIA calculates the Doppler frequency shift and the haemodynamic variables flow, volume, and velocity of each pixel. The resulting perfusion image was processed with respect to (1) underexposed and overexposed pixels, (2) saccades, and (3) the retinal vessel tree. The rim area and the saccades were marked interactively by the operator. The capillaries and vessels of the retinal vessel tree were identified automatically by pattern analysis. Retinal vessels with a diameter greater than 30 microns, underexposed or overexposed areas, and saccades were excluded automatically. Based on the whole perfusion image total mean flow, total mean volume, total mean velocity, standard deviation, cumulative distribution curve of flow, and the capillary pulsation index were calculated automatically. Heart beat associated pulsation of capillary blood flow was estimated by plotting the mean capillary flow of each horizontal line against time. Intraobserver reliability was estimated by measuring 10 eyes of 10 subjects on five different days by one observer. Interobserver reliability of AFFPIA was evaluated by analysing 10 perfusion maps by five different operators. To find a baseline of retinal blood flow, perfusion maps of 67 eyes of normal subjects with a mean age of 40.4 (SD 15) years were evaluated by AFFPIA. RESULTS: The coefficient of reliability of the intraobserver reproducibility of flow was 0.74. The coefficient of reliability of the interobserver reproducibility was 0.95. The juxtapapillary retinal capillary flow was temporally 484 (SD 125), nasally 450 (117); the rim area capillary flow was 443 (110). The mean capillary pulsation index of retinal flow was 0.56 (0.14). CONCLUSION: Retinal blood flow evaluation by the AFFPIA increases significantly the interobserver reliability compared with conventional evaluation of 100 microns x 100 microns areas in SLDF images with the original Heidelberg retina flowmeter software. The intraobserver reliability of AFFPIA was in the same range as conventional evaluation.