Vascular Niche Changes And Its Influnence On HSC/HPCs In GAHD Mice

IntroductionAllogeneic hematopoietic stem cell transplantation (allo-HSCT) has beenaccepted as the most effective treatment strategy for hematological malignancies,benign hematological disorders, solid tumors, inherited diseases and some otherautoimmune diseases. Acute graft-versus-host disease (aGVHD) is the most commoncomplication which oftern affects the outcome of allo-HSCT. Pathophysiologicalmechanisms of aGVHD are that the immunocompetent cells from donor (Tlymphocytes) directly or indirectly lead to target organ damage. The impairedhematopoiesis in transplant recipients that develop aGVHD has been clearlydemonstrated. The recipients with impaired hematopoiesis post-transplanatation dueto GVHD often have a poor prognosis. The mechanisms for hematopoieitcdysfunction after aGVHD are not clear. Now at least two mechanisms have beeensuggersted:(1) a hematopoietic inhibition mediated by inhibitory cytokines, such asTNF-a, produced during this disorder as part of a "cytokine storm";(2) a hibition dueto a deficient bone marrow microenvironment (niche) damaged by the conditioning bythe cytokines produced. In transpalant recipients, the hematopoietic system is derivedfrom the dinor, thus, it is not regarged as a major target for aGVHD; but the recipent'sbone marrow niche remains to be from host, so in theory. the host's bone marrowniche could be the target of aGVHD. Two types of bone amrrow niche were defined inrecent years. One type of niche is named endosteal niche, which is mainly located inthe endosteal and formed with osteoblast; the other is named vascular niche, which isformed with sinusoidal vascular endothelial cells and perivascular cells. It is has beendemonstrated that Vascular niche plays an important role in regualtion self-renewalcapacity and maintaining the stablity of HSC pool. The major targets of aGVHD areliver, skin and interstinal tract, all of which are characterized by being covered withendothelial cells. So we suggested that vascular niche might be the target of aGVHDand its dysfuction was the immportant reason of impared hematopoiesis post aGVHD.To clarify the dysfunction of vascular niche and its effects on the biologicalcharacteristics of HSCs, we proceeded this study to address the following main issues:1.How to identify the murine bone marrow sinusoidal endothelial cells? Theendothelial cells from bone marrow were sorted with multi-parameter flowcytometry with antibodies and identified with cytochemistry staining and other means. Wedeveloped MHC haploidentical matched aGVHD mouse model.2. What are themechanisms of vascular niche dysfunction in aGVHD condition? Does donor T cellsthrough Fas/FasL pathway cause apoptosis of BM endothelial cells? The numbers ofbone marrow vascular endothelial cells from aGVHD mouse and controls weredetected with Flow cytometry, pathological section and RT-PCR was used to analysethe ratio of proliferation, apoptosis, MHC molecule and FasL expressioin in T cellsubsets.3.Under the condition of aGVHD, the effects of vascular niche dysfuction onHSCs/HPCs. The SLAM ratio of the stem cells were detected with Flow cytometry;the self-renewal and differentiation capacity of HSCs were analysed with competitivetransplantation and serial transplantation tests. The expression of chemokine CXCR4in HSCs and SDF-1in endothelial cells (SDF-1/CXCR4axis were analysed withFlow cytometry; the gene expression level of C-Kit in stem cells and SCF (SCF/C-Kitaxis) in bone marrow endothelial cells were detected with RT-PCR,.Part ⅠEstablishment of haploidentical matched aGVHD mouse model andidentification of bone marrow sinusoidal endothelial cellsMethods:Establishment of haploidentical matched aGVHD mouse modelDonor bone marrow cells were from Balb/C(CD45.2; H-2d) and host bonemarrow cells were from CB6F1(Balb/C CD45.2; H-2d×C57BL/6CD45.1H-2b；F1:CD45.1/2H-2b/d) mice respectively. The study were designed with3groups. GVHDgroup: Each receptor was transplanted with F1mouse bone marrow cells of5×106and spleen cells of6×107. BMT group: Each receptor was transplanted with only F1mouse bone marrow cells of5×106. PBS group: Each receptor was transplanted withonly PBS in same volume. Each receptor received the irradiation with a dosage of8Gy isotope cesium on one day before transplantation. The degree of aGVHD andsurvival of recipient mice were identified. Liver, skin and small intestine were takenfrom GVHD and BMT group mice to confirmed aGVHD with histopathology at14days after transplantation. CD45.1and CD45.2expression of hematopoietic cells weredetected with Flow cytometry to define the donor cell engraftment.Identification of Bone marrow vascular sinusoidal endothelial cell phenotypeSca-1, CD45, VEGFR2, VEGFR3expression of mononuclear cells (MNCs) from normal mouse bone marrow was detected with flow cytometry.VEGFR2+/VEGFR3/+Sca-1-cells were sorted with FCM for VE-cadherin staining.The positive rate was observed with fluorescence microscopy. Sorted CD4-/CD8+Tcells were as controls.Statistical analysis: results expressed as mean±standard deviation.two-samplemean comparison using the Student's test. Survival using the Kaplan-Meier survivalanalysis.ResultsaGVHD mouse model was successfully established. Mice in PBS group began todie at10days after transplantation. VEGFR3was the most specific markers. Flowcytometry SSC/VEGFR3gating can be used in further studies for bone marrowendothelial cells analysis.part Ⅱ The changes of bone marrow vascular endothelial cells inaGVHD and its relationship to the hematopoietic stem cellsMethodsSecond transplantation was used to detect the hematopoietic stem cellscapacity of proliferation and differentiation after first transplantationContinuous transplantation14days after first transplantation, GVHD andBMT group mice (donor: CB6F1H-2b/d, phenotype CD45.1/2; recipient: Balb/C H-2dphenotype CD45.2) were killed, MNCs were got from the two groups, Number of5×106/per-mouse cells implanted into healthy Balb/C recipients and8Gy radiotherapybefore transplantation.14days after the second transplantation, flow cytometry wasused to detect the quantity of bone marrow mononuclear and ratio of myeloid (Gr-1+),mononuclear (CD11b+), B cells (B220+).Competitive transplantation14days after the first transplantation, thetransplanted mice (donor: CB6F1H-2b/dphenotype CD45.1/2; recipient:Balb/C H-2d,phenotype CD45.2) were killed. PBS flushed to get the MNC, and mixed with equalamount of MNC which come from healthy F1mice (CB6F1H-2b/d, phenotypeCD45.2). Total number of5×106cells implanted into Balb/C recipients after8Gyradiotherapy. Flow cytometry was used to detect CD45.1+/CD45.2+hematopoieticcells and the ratio of B myeloid (Gr-1+), mononuclear (CD11b+), cells (B220+). The ratio of hematopoietic stem cells detected by flow cytometry in mousebone marrow14and21days after transplantation in of GVHD group and BMTgroups, the ratio of Lin-/CD48-/CD150+cells to MNC was detected. CXCR4+expression in HSCs and SDF-1+in SECs was detected too (SDF-1/CXCR4axis).RT-PCR was used to detect the relative expression of SCF in SECs and C-kitin HSCs (SCF/C-kit axis)14days after transplantation, Bone marrowmononuclear cells were got from each group, VEGFR2+/VEGFR3+/Sca-1-gated tosort bone marrow endothelial cells, the number of5-8×105lin-/CD48-/CD150+hematopoietic stem cells was taken, Trizol cracked and frozen at refrigerator.Statistical analysis results expressed as mean±standard deviation or percentage,two-sample mean comparison using the Student's test.ResultsContinuous transplantation14days after transplantation, MNC count/per-tibia, the ratio of granulocytes (Gr-1+), monocytes(CD11b+), B cells (B220+) inMNC and the absolute count showed no difference between the two groups(P>0.05).Competitive transplantation14days after transplantation, MNC count/per-tibia and CD45.1+/CD45.2+absolute value and the ratio of granulocytes (Gr-1+),the monocytes(CD11b+), the ratio of B cells (B220+) showed no difference.(P>0.05).The ratio of HSCs to MNCs in GVHD is lower than the BMT group14daysafter transplantation In GVHD group, the ratio of Lin-/CD48-/CD150+to MNCswas (0.6175±0.033)%, the ratio was (0.745±0.015)%in BMT group(P=0.013).CXCR4expression of HSCs showed no difference14days aftertransplantation between the two groups CXCR4in Lin-/CD48-/CD150+cellswas high expressed in two groups, the ratio of GVHD group was (96.5±7.65)%.andthe ratio was (94.8±6.42)%in BMT (P>0.05).SDF-1expression of SECs showed no difference14days after transplantationbetween the two groups (P>0.05) The ratio in GVHD group was (93.7±8.53)%,BMT was (87.5±10.61)%.SCF expression of bone marrow endothelial cells in GVHD is Lower than theBMT group by RT-PCR14days after transplantation Relative expression inGVHD was:0.0326±0.004, the expression was:0.3285±0.079in BMT group.C-Kit expression of hematopoietic stem cells in GVHD14days aftertransplantation RT-PCR was used to detect the C-Kit relative expression in HSCsbetween the two groups, The relative expression in GVHD was0.0205±0.002, the relative expression in BMT group was0.0341±0.003.ConclusionThe above results showed: the number of HSCs was lower than the BMT groupunder the condition of GVHD, but continuous transplantation and competitivetransplantation proved that HSCs derived from GVHD mice whose capacity ofproliferation and differentiation is not lower than the BMT group, so the inhibitionfactor came from stromal cells. Endothelial cells did not affect the stem cells throughthe SDF-1/CXCR4axis. SCF relativeexpression in bone marrow sinosiodalendothelial cells was lower than the BMT group, Bone marrow SECs damage wasfrom the microenvironment, but not hematopoietic stem cells itselfs. Bone marrowSECs probably affect stem cell proliferation through the SCF/C-Kit passway.Part Ⅲ The changes of mice bone marrow sinusoidal endothelial cellsand donor T cells in acute GVHDMethodsDetected the number of mouse bone marrow SECs, the ratio of proliferationand MHC molecules, and Fas expression in SECs by flow cytometry.Single tibia was repeatedly flushed by PBS to get mononuclear cells in each groupat+14and21days. Counted under light microscopycounted and lysised red bloodcells for flow cytometry. Make sure the number and the ratio of SECs to MNCs.Detected the VEGFR2+/VEGFR3+/Sca-1-ratio with FCM. Combined with themononuclear cell count, we calculated the absolute value of SECs. ki-67was used forSECs proliferation. Annexin V+/PI-was used for apoptosis and Fas, MHC-I, MHC-IIwas for Fas and MHC molecule expression detection.Detected the ratio of T cell subsets, and the source and expression of FasL14and21days after transplantation, CD8/CD4was gated for T cell subsets, anddetected the intensity of FasL expression in CD8or CD4+T cells. CD45.1/CD45.2gated to detect the source of T cells.Pathologically detected the degree of proliferation and changes in SECs14and21days after transoplantation, the routine HE and immunohistochemicalstaining and plastic-embeded sections was made, then observed under microscopy.ELISA method was used to measure the concentration of endothelial growthfactor.14,21days after transplantation, single tibia of GVHD and BMT group micerepeatedly flushed by constant volume of1ml PBS, collected the supernatant, conventionally took the serum, VEGF, SDF-1, bFGF concentration in the serum andsupernatant was detected with ELISA Kit.Detected apoptosis-related Fas and caspase-3genes expression in bonemarrow SECs with RT-PCR14days after transplantation, VEGFR2+/VEGFR3+/Sca-1–gated to sort the number of5-8×105SECs, CD8/CD4gated to sort the numberof106CD4/CD8-and CD4-/CD8+T cells, Trizol percussed, then kept at-40°C, andcustomized the relevant primers for next step.Statistical analysis results expressed as mean±standard deviation, two-samplemean comparison using the Student's test.Results14,21days after transplantation, MNC count in each tibia of the GVHDwere lower than the BMT group (P=0.0316, P=0.0076) MNC count in GVHDon+14,21d was respectively (1.10±0.21)×107and (0.58±0.14)×107, MNC count inBMT group was respectively (1.71±0.32)×107,(2.14±0.38)×107.14,21days after transplantation, the ratio of SECs to MNC in GVHD wasbelow the BMT group (P=0.0253, P=0.0017) The ratio was respectively(0.145±0.132)%,(0.028±0.019)%in GVHD group at+14,+21d, and BMT wasrespectively (0.335±0.070)%,(0.552±0.124)%.14,21days after transplantation, SECs in each tibia, absolute numbers inGVHD were lower than the BMT group (N=4,P=0.0037,P=0.0001)14,21daysafter transplantation, SECs count in each tibia of GVHD group was respectively(1.595±0.41)×104,(0.3234±0.11)×104. The BMT group count was respectively(5.695±0.78)×104,(11.55±2.38)×104.14days after transplantation, Ki-67expression in bone marrow SECsshowed no difference between two groups (P=0.053) The ratio of VEGFR3+/Ki-67+cells to MNC respectively (0.125±0.053)%,(0.275±0.058)%14,21days after transplantation, apoptosis of SECs increased in GVHD (P=0.016, P=0.010)14and21days after transplantation, the ratio of Annexin V+/PI-to SSClow/VEGFR3+was respectively (39.57±2.77)%,(29.87±2.55)%in GVHDgroup.The ratio was respectively (28.37±1.97)%,(18.47±1.72)%in the BMT group.14,21days after transplantation, the ratio of MHC-I molecules expression inbone marrow SECs in GVHD show no difference with BMT group (P=0.470, P=0.372) MHC-I+ratio in SSClow/VEGFR3+was respectively (81.62±3.64)%,(70.10±3.19)%. The ratio of the BMT group was respectively (78.35±3.64)%, (64.27±5.13)%.14,21days after transplantation, the ratio of MHC-II positive expression inbone marrow SECs in GVHD is higher than the BMT group (P=0.00035, P=0.033) The positive expression of MHC-II in SSClow/VEGFR3+cells wasrespectively (57.32±3.61)%,(15.60±1.25)%. The BMT group was respectively(21.25±3.09)%,(9.95±2.68)%.14,21days after transplantation, The ratio of Fas+expression in GVHD bonemarrow SECs is higher than the BMT group (p=0.0036, P=0.0011) ratio ofFas+expression in SSC low expression/VEGFR3+endothelial cells in GVHD groupwas respectively (87.6±3.48)%,(72.5±3.33)%, the ratio of the BMT group wasrespectively (63.92±3.76)%,(26.55±7.17)%.14,21days after transplantation, the ratio of FasL expression in GVHDCD4+T cells were higher than the BMT group (P=0.007, P=0.0004) The ratioof Fas expression in CD4+/CD8-T cells in GVHD group was respectively (72.8±6.24)%,(72.2±4.31)%. The ratio of the BMT group was respectively (45.72±2.53)%,(31.35±7.84)%.14,21days after transplantation, the ratio of FasL expression in GVHD CD8+T cells were higher than the BMT group (P=0.002, P=0.043) The ratio of Fasexpression in CD8+T cells in GVHD was respectively (56.32±3.99)%,(22.31±4.75)%in GVHD group. the ratio of the BMT group was respectively (15.95±3.27)%,(9.82±1.11)%.14days after transplantation, GVHD and BMT bone marrow CD4+T cellswere from the donors The ratio of CD45.1+/CD45.2+to CD4+T cells wasrespectively (97.96±0.76)%,(96.57±1.10)%.7,14and21days after transplantation, bone marrow hyperplasia andchanges of vascular endothelial cell were detected by pathological methods inGVHD and BMT groups.Morphological identification of endothelial cells were detected with HE,immunohistochemical staining and plastic-embedded sections, which characterized bydistributed along the sinusoids with the narrow cells and spindle-shaped nuclei.Immunohistochemically the VEGFR3+endothelial cell showed brown cytoplasmnuclear. Plastic-embedded sections from the primitive to the mature stage of cellcytoplasm were from slight to deep blue, Thus the sinusoids and endothelial cellscould be identified clearly. Bone marrow of normal mouse actively proliferated, flat-like sinusoids could beseen, no mature red blood cells spills outside sinusoids.Bone marrow of PBS group hyperplasia was inhibited, there are rare cells,mainly vacuoles, almost no sinusoids and endothelial cells were seen14days aftertransplantation.Bone marrow hyperplasia was inhibited in GVHD group14days aftertransplantation, the nucleated cells was significantly reduced, vacuolar can be seemore common, the number of sinusoids decreased significantly, sinuses swelled,closed-construction destroyed, and the mature red blood cells escape outside thesinusoids. At+21d the nucleated cells is further reduced, and endothelial cells wereseen occasionally, sinusoid structure is hardly recognizable.14days after transplantation, more active hyperplasia was observed in BMT group,nucleated cells were more common, sinusoid damage mitigated than the GVHD group,Some mature red blood cells escaped out of the sinusoids.21days aftertransplantation, proliferation was active, sinusoids shape recovery, structuralintegrized, and no red blood cells were seen to escape.VEGF, SDF-1, bFGF concentration of serum and bone marrow supernatantwere detected by ELISA Kit in two groups7,14,21days after transplantation, no significant difference was found inVEGF concentration of the bone marrow supernatant between the two groups(P>0.05) VEGF concentration in the GVHD group was respectively (32.84±8.76)ng/ml,(55.64±9.23) ng/ml,(22.19±5.41) ng/ml. The BMT group was respectively(51.08±13.18) ng/ml,(125.70±22.09) ng/ml,(50.17±13.47) ng/ml.7,14days after transplantation, the serum concentration of VEGF in GVHDgroup was lower than the BMT group (P=0.015; P=0.025).21days aftertransplantation, no significant difference was found between the two groups (P=0.732) In the GVHD group the serum concentration was respectively (125.18±4.68)ng/ml,(149.16±4.71) ng/ml,(179.62±45.23) ng/ml. In the BMT group wasrespectively (153.52±5.17) ng/ml,(229.92±3.84) ng/ml,(162.55±21.74) ng/ml.7,14,21days after transplantation, no significant difference was found inSDF-1concentration of supernatants between the two groups (P>0.05)Concentration of supernatants in GVHD group was respectively (164.52±32.14)ng/ml,(156.85±32.68) ng/ml,(139.80±26.47) ng/ml, In BMT group was respectively(178.88±23.56) ng/ml,(196.38±30.65) ng/ml,(154.33±45.58) ng/ml. 7,14,21days after transplantation, no significant difference was found inserum concentration of SDF-1between the two groups(P>0.05) concentration ofSDF-1in GVHD group was respectively (113.78±30.13)ng/ml,(395.46±236.30)ng/ml,(278.80±97.90) ng/ml. The BMT group was respectively (217.65±76.18)ng/ml(563.67±146.25) ng/ml,(486.16±22.78) ng/ml.7,14,21days after transplantation, no significant difference was found inSupernatant concentration of bFGF between the two groups (P>0.05)Supernatant concentration in GVHD was respectively (181.825±47.70) ng/ml,(168.90±39.21) ng/ml,(104.07±16.77)ng/ml. The BMT group was respectively(234.325±44.37) ng/ml,(127.62±32.15) ng/ml.7,14,21days after transplantation, no significant difference was found in Serum concentration of bFGF between the two groups (P>0.05) GVHD in thegroup was respectively (119.57±13.49)ng/ml,(83.58±14.04) ng/ml,(222.34±73.07)ng ml. The BMT group was respectively (110.8±17.44) ng/ml,(211.03±40.08) ng/ml,(111.44±17.93) ng/ml.Relative expression of the Fas, Caspase-3gene by RT-PCR14days after transplantation, RT-PCR method was used to detect the expressionof Fas and Caspase-3in bone marrow endothelial cell. Gene expression in GVHDwas higher than the BMT group: relative expression of the Fas gene in GVHD groupwas0.2536±0.021, the BMT group relative expression was0.0852±0.013. Caspase-3gene relative expression in GVHD group was0.2910±0.086, the BMT group was:0.0323±0.013.ConclusionThese experiments confirmed that GVHD bone marrow MNC was reducedcompared with BMT group. The ratio and the number of endothelial cells decreased;FasL expression increased in donor CD4+and CD8+T cells. High Fas expression wasfound in endothelial cells, experiments showed that SECs increased in apoptosis.Proliferation did not change significantly, angiogenic growth factors showed nodifference between two groups except serum VEGF. Fas, caspase-3expression inBone marrow SECs increased in GVHD, endothelial cells express MHC-II moleculesmainly, suggesting that bone marrow endothelial cell apoptosis was caused by CD4+T cells through the Fas/FasL pathway.