Noninvasive prenatal paternity testing by means of SNP-based targeted sequencing.

OBJECTIVE: To develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs). METHOD: SNPs were selected based on population genetics data. Target-SNPs in cell-free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated. RESULTS: Fetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid-derived fetal genomic DNA. From an initial panel of 356 target-SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short-tandem-repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men. CONCLUSION: The novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target-SNPs, increased accuracy, and reduced costs.

Whole extracts of Radix Achyranthis Bidentatae and Radix Cyathulae promote angiogenesis in human umbilical vein endothelial cells in vitro and in zebrafish in vivo.

Although Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) are from two different medicinal plants, they are both used as 'Niu-Xi', a widely used traditional Chinese medicine that is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. The aim of the present study was to investigate whether the whole extracts of RAB and RC possess pro-angiogenic effects. In order to examine this idea whole extracts of RAB and RC were extracted with boiling water followed by ethanol, respectively. Results from the MTT, wound healing and tube formation assays in human umbilical vein endothelial cells (HUVECs) in vitro revealed that the whole extracts of RAB and RC did not increase cell proliferation or tube formation, but enhanced cell migration. Their angiogenic effects were also confirmed in zebrafish in vivo via increasing the sprout numbers in the sub-intestinal vessel. As determined by quantitative polymerase chain reaction, the whole extracts of RAB and RC both regulated the expression of cell migration-related genes in zebrafish. It is concluded that the whole extracts of RAB and RC induced angiogenesis in HUVECs in vitro and in zebrafish in vivo via increasing cell migration.

Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula.

Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

Enumeration and functional investigation of endothelial progenitor cells in neovascularization of diabetic foot ulcer rats with a Chinese 2-herb formula.

BACKBROUND: We investigated the effect of a Chinese 2-herb formula (NF3) on the enumeration and angiogenic differentiation of endothelial progenitor cells (EPCs) in diabetic foot ulcer rats. METHODS: EPCs and stromal cell-derived factor-1α (SDF-1α) were quantified by flow cytometry and ELISA, respectively. In vitro angiogenesis assays included proliferation, adhesion, migration and tube formation. RESULTS: Our result demonstrated that NF3 (0.98 g/kg) could significantly enhance the circulating CD34(+) /VEGFR2(+) /CD45(-) EPCs levels in diabetic foot ulcer rats by 60% (P < 0.05) through the partial elevation of SDF-1α, restoring the mobilization ability of EPCs for wound neovascularization. We successfully isolated the BM-derived EPCs to study their angiogenic potential after NF3 treatment. BM-derived EPCs significantly expressed cell surface markers of CD34, CD146 and VEGFR2 (P < 0.05 - 0.01). NF3 could significantly stimulate the proliferation and attachment ability of EPCs dose-dependently (P < 0.01-0.001). Besides, NF3 could significantly augment EPCs migration (P < 0.001) and tube formation (P < 0.01-0.001). CONCLUSIONS: NF3 modulated diabetic wound healing through regulation of systemic EPCs level and increase in local vascular formation.

Comprehensive proteomic analysis of a Chinese 2-herb formula (Astragali Radix and Rehmanniae Radix) on mature endothelial cells.

Endothelial cells are crucially involved in wound healing angiogenesis, restoring blood flow to wound tissues. Our previous study demonstrated that the Chinese 2-herb formula (NF3) possesses significant wound healing effect in diabetic foot ulcer rats with promising in vitro proangiogenic effects on human umbilical vein endothelial cells (HUVEC). Here, we present the comparative global proteome analysis of NF3-treated HUVEC in static or scratch conditions, screening the comprehensive molecular targets in governing the proangiogenic response in wound healing. Our results suggest plasminogen activator inhibitor-1, specifically down-regulated in static condition and Annexin A1 and Annexin A2, up-regulated in scratch condition, as principal proteins responsible for the proangiogenesis in wound healing. We also identified a panel of cytoskeleton regulatory proteins in static and scratch condition, mediating the migratory behavior of NF3-treated HUVEC. The key proteins in static state include myosin regulatory light polypeptide 9, SPAST, tropomyosin (TPM)2, and Vimentin while that in scratch state contained prelamin-A/C, TPM1, TPM2, and Vimentin. In addition, NF3 was shown to regulate transcription and translation, cell-cell interaction, and ROS defense in HUVEC. Proliferation and migration assays further confirmed the identified principal proteins plasminogen activator inhibitor-1 and Annexin A2 which are responsible for NF3-induced proangiogenesis of HUVEC in wound healing. This is the first study on the global proteome expression of NF3-treated HUVEC with the identification of the differences at the molecular level, between static and scratch conditions involved in wound healing angiogenesis.

A Chinese 2-herb formula (NF3) promotes hindlimb ischemia-induced neovascularization and wound healing of diabetic rats.

Diabetic foot ulcer is closely associated with peripheral vascular disease. Enhancement of tissue oxidative stress, reduction of nitric oxide (NO) and angiogenic growth factors, and abnormal matrix metalloproteinase (MMP) activity are pathophysiological factors in post-ischemic neovascularization and diabetic wound healing. Our previous study demonstrated that the Chinese 2-herb formula, NF3, showed significant wound healing effects on diabetic foot ulcer rats. A novel rat diabetic foot ulcer with hindlimb ischemia model was established in order to strengthen our claims on the diabetic wound healing and post-ischemic neovascularization effects of NF3. Our results demonstrate that NF3 can significantly reduce the wound area of the diabetic foot ulcer rat with hindlimb ischemia by 21.6% (p<0.05) compared with the control group. In addition, flow cytometric analysis revealed that NF3 could boost circulating EPC levels for local wound vessel incorporation. Immunohistochemical analysis showed that NF3 could significantly augment blood vessel density, VEGF and eNOS expression, and attenuate tissue oxidative stress of ischemic muscles (p<0.001). NF3 significantly stimulated MMP activity involved in angiogenesis. Our study shows, for the first time, the beneficial effects of NF3 in wound healing and post-ischemic neovascularization in diabetes.

In vitro and in vivo mechanistic study of a novel proanthocyanidin, GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis.

Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (P<.001) and tubule formation (P<.001-.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC-(4→8)-GCG in a dose-dependent manner after 3 days of treatment (P<.001-.05). In conclusion, these results revealed that our novel proanthocyanidin, GC-(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent.

Gene expression profiling on the molecular action of danshen-gegen formula in a randomized placebo-controlled trial of postmenopausal women with hypercholesterolemia.

The Danshen-Gegen formula (DG) is a traditional Chinese herbal formula which has long been used to treat cardiovascular disease. DG was found to be a cardiovascular tonic in our recent research. However, a comprehensive investigation of the molecular mechanism of DG in cardiovascular disease has not been performed. The aim of this study was to clarify the transcriptional profiling of genes modulated by DG on postmenopausal women by using DNAmicroarray technology. We obtained 29 whole blood samples both from DG-treated and placebo-treated subjects. Blood lipid profile and intima-media thickness (IMT) were measured. Affymetrix GeneChip was used to identify differentially expressed genes (DEGs), followed by validation by the real-time PCR method. The results showed that DG-treated group has a significant improvement in IMT and lipid profile as compared to placebo-treated group. For the genomic study, the DG-treated group has a higher number of DEGs identified as compared to the placebo-treated group. Two important biological processes of "regulation of systemic arterial blood pressure by hormone" and "regulation of smooth muscle proliferation" have been identified by GePS in the DG-treated group. No significant biological process and cellular components were identified in the placebo-treated group. This genomic study on the molecular action of DG in postmenopausal women gathered sufficient molecular targets and pathways to reveal that DG could improve neointima thickening and hypertension.

Molecular angiogenic events of a two-herb wound healing formula involving MAPK and Akt signaling pathways in human vascular endothelial cells.

The emergence of electric cell-substrate impedance sensing (ECIS) technology has provided new insight in advanced cell behavioral study by its nanometer sensitivity, precise electrical wounds generation, and high reproducibility that can be monitored in real time in a noninvasive way. However, little is known regarding pro-angiogenic agents in wound healing studies using endothelial cells evaluated with ECIS technology. Our previous studies showed a prominent wound healing effect of a two-herb formula (NF3) comprising of Astragali Radix and Rehmanniae Radix in a rat chronic wound model through actions including angiogenesis. Here we further investigated the angiogenic effect and its underlying molecular mechanism through proliferation, motility, and tubule formation of human vascular endothelial cells (HECV) using ECIS technology. It was first shown that HECV treated with NF3 had a higher resistance than that of control using ECIS cell attachment and cell migration model (p < 0.01). We further validated in a scratch assay that NF3 treatment significantly stimulated HECV cell migration (p < 0.01-0.05). Also, NF3-treated HECV were observed to develop into a significantly more branched tubular structure when compared with control (p < 0.05-0.01). Meanwhile, Western blot analysis of NF3-treated HECV revealed the activated expression of p-Akt, and mitogen-activated protein (MAP) kinases for p-ERK, p-p38, and p-JNK. We propose that the effect of NF3 in the promotion of endothelial cell migration and tubule formation could be mediated through pathways involving p-Akt and activated MAP kinases. Hence, we demonstrated the complexity of the angiogenic effect activated by NF3 molecularly and functionally. NF3 treatment could offer therapeutic value to chronic wound healing for its pro-angiogenic efficacy.

In Vivo Study on the Pharmacological Interactions between a Chinese Herbal Formula ELP and Antiresorptive Drugs to Counteract Osteoporosis.

Antiresorptive drugs, alendronate and raloxifene, are effective in lowering bone mineral density (BMD) loss in postmenopausal women. However, long-term treatment may be associated with serious side effects. Our research group has recently discovered that a Chinese herbal formula, ELP, could significantly reduce BMD loss in animal and human studies. Therefore, the present study aimed to investigate the potential synergistic bone-protective effects of different herb-drug combinations using ovariectomized rats. To assess the efficacy of different combinations, the total BMD was monitored biweekly in the 8-week course of daily oral treatment. Bone microarchitecture, bone strength, and deoxypyridinoline level were also determined after 8 weeks. From our results, coadministration of ELP and raloxifene increased the total tibial BMD by 5.26% (2.5 mg/kg/day of raloxifene; P = 0.014) and 5.94% (0.25 mg/kg/day of raloxifene; P = 0.026) when compared with the respective dosage groups with raloxifene alone. Similar synergistic effects were also observed in BMD increase at distal femur (0.25 mg/kg/day; P = 0.001) and reduction in urinary deoxypyridinoline crosslink excretion (2.5 and 0.25 mg/kg/day; both P = 0.02). However, such interactions could not be observed in all alendronate-treated groups. Our data provide first evidence that ELP could synergistically enhance the therapeutic effects of raloxifene, so that the clinical dosage of raloxifene could be reduced.

Synergistic interaction between Astragali Radix and Rehmanniae Radix in a Chinese herbal formula to promote diabetic wound healing.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali Radix (AR) and Rehmanniae Radix (RR) are two traditional Chinese medicines widely used in China for treating diabetes mellitus and its complications, such as diabetic foot ulcer. AIM OF STUDY: In our previous study, a herbal formula NF3 comprising AR and RR in the ratio of 2:1 was found effective in enhancing diabetic wound healing in rats through the actions of tissue regeneration, angiogenesis promotion and inflammation inhibition. The aims of the present study were to investigate the herb-herb interaction (or the possible synergistic effect) between AR and RR in NF3 to promote diabetic wound healing and to identify the principal herb in the formula by evaluating the potencies of individual AR and RR in different mechanistic studies. MATERIALS AND METHODS: A chemically induced diabetic foot ulcer rat model was used to examine the wound healing effect of NF3 and its individual herbs AR and RR. For mechanistic studies, murine macrophage cell (RAW 264.7) inflammation, human fibroblast (Hs27) proliferation and human endothelial cell (HMEC-1) migration assays were adopted to investigate the anti-inflammatory, granulation formation and angiogenesis-promoting activities of the herbal extracts, respectively. RESULTS: In the foot ulcer animal model, neither AR nor RR at clinical relevant dose (0.98g/kg) promoted diabetic wound healing. However, when they were used in combination as NF3, synergistic interaction was demonstrated, of which NF3 could significantly reduce the wound area of rats when compared to water group (p<0.01). For anti-inflammation and granulation formation, AR was more effective than RR in inhibiting lipopolysaccharide (LPS)-induced nitric oxide production from RAW 264.7 cells and promoting Hs27 fibroblast proliferation. In the aspect of angiogenesis promotion, only NF3 promoted cell migration of HMEC-1 cells. CONCLUSIONS: AR plays a preeminent role in the anti-inflammatory and fibroblast-proliferating activities of NF3. The inclusion of RR, however, is crucial for NF3 to exert its overall wound-healing as well as the underlying angiogenesis-promoting effects. The results of present study justified the combined usage of AR and RR in the ratio of 2:1 as NF3 to treat diabetic foot ulcer and illustrated that AR is the principal herb in this herbal formula.

The in vivo and in vitro diabetic wound healing effects of a 2-herb formula and its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs Radix Astragali (RA) and Radix Rehmanniae (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. AIM OF STUDY: Diabetic complications, such as foot ulcer, impose major public health burdens worldwide. In our previous clinical studies, two Chinese medicine formulae F1 and F2 have achieved over 80% limb salvage. A simplified 2-herb formula (NF3) comprising of RA and RR in the ratio of 2:1 was used for further study. NF3 was examined for the ulcer healing effect in diabetic rats, and its potential mechanisms of action in fibroblast proliferation, angiogenesis and anti-inflammation in vitro. MATERIALS AND METHODS: A chemically induced diabetic foot ulcer rat model was used for studying the wound healing effect. In the in vitro mechanistic studies, human fibroblast cells (Hs27), human umbilical vein endothelial cells (HUVEC) and mouse macrophage cells (RAW264.7) were assessed for tissue regeneration, angiogenesis and anti-inflammatory activities, respectively. RESULTS: Our in vivo results demonstrated a significant reduction of wound area at day 8 in NF3 (0.98g/kg) group as compared to control (p<0.01). NF3 could significantly stimulate Hs27 proliferation in a dose dependent manner (p<0.05). Besides, NF3 could significantly increase the cell migration and tube formation (p<0.05-0.001) of HUVEC in the angiogenesis study. Furthermore, significant inhibition of nitric oxide production (p<0.01) was found in NF3-treated macrophage cells, suggesting its anti-inflammatory activity. CONCLUSIONS: Our study presents for the first time scientific evidence towards the efficacy of the two-herb formula NF3 in enhancing diabetic wound healing through the actions of tissue regeneration, angiogenesis and anti-inflammation.