Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells.

Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers--CD34, neuropilin 1, and human kinase insert domain-containing receptor--and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice.

UNLABELLED: Sorafenib--a broad kinase inhibitor--is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia--and its consequences on treatment resistance--remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen-positive (Gr-1(+)) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1(+) myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis--despite persistently elevated hypoxia--in part by reducing Gr-1(+) myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. CONCLUSION: Blocking SDF-1α/CXCR4 or Gr-1(+) myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment.

Mindin (SPON2) Is Essential for Cutaneous Fibrogenesis in a Mouse Model of Systemic Sclerosis.

Systemic sclerosis is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as systemic sclerosis has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor SNAI1 is overexpressed in the epidermis of patients with systemic sclerosis, and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (SPON2) in fibrogenesis. Mindin is produced by SNAI1 transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Polymeric micelles effectively reprogram the tumor microenvironment to potentiate nano-immunotherapy in mouse breast cancer models.

Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.

Normalizing the Microenvironment Overcomes Vessel Compression and Resistance to Nano-immunotherapy in Breast Cancer Lung Metastasis.

Nano-immunotherapy regimens have high potential to improve patient outcomes, as already demonstrated in advanced triple negative breast cancer with nanoparticle albumin-bound paclitaxel and the immune checkpoint blocker (ICB) atezolizumab. This regimen, however, does not lead to cures with median survival lasting less than two years. Thus, understanding the mechanisms of resistance to and development of strategies to enhance nano-immunotherapy in breast cancer are urgently needed. Here, in human tissue it is shown that blood vessels in breast cancer lung metastases are compressed leading to hypoxia. This pathophysiology exists in murine spontaneous models of triple negative breast cancer lung metastases, along with low levels of perfusion. Because this pathophysiology is consistent with elevated levels of solid stress, the mechanotherapeutic tranilast, which decompressed lung metastasis vessels, is administered to mice bearing metastases, thereby restoring perfusion and alleviating hypoxia. As a result, the nanomedicine Doxil causes cytotoxic effects into metastases more efficiently, stimulating anti-tumor immunity. Indeed, when combining tranilast with Doxil and ICBs, synergistic effects on efficacy, with all mice cured in one of the two ICB-insensitive tumor models investigated is resulted. These results suggest that strategies to treat breast cancer with nano-immunotherapy should also include a mechanotherapeutic to decompress vessels.

PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Targeted delivery of AAV-transduced mesenchymal stromal cells to hepatic tissue for ex vivo gene therapy.

Adeno-associated virus (AAV)-mediated gene therapy holds great promise if challenges related to vector neutralization by pre-existing antibodies are circumvented. The use of autologous or allogeneic cells to shield the vector might offer the possibility of successful gene transfer in such a situation. In the present study, we evaluated the feasibility of AAV-transduced mesenchymal stromal cells (MSCs) as a vehicle for hepatic gene transfer in a murine liver injury model. In our initial studies to determine the most suitable vector, we observed that AAV1 (91%) and AAV6 (72%) serotypes are highly efficient in transducing MSCs. Subsequently, we generated a transient liver injury model to analyse the efficacy of MSCs homing to the liver, as well as their hepatic gene transfer efficiency; our data show that administration of acetaminophen (500 mg/kg) served as a cue for the homing of MSCs to the liver. Furthermore, sex-mismatched transplantation of AAV1-infected MSCs demonstrated a 3.5-fold (day 7) and 2.2-fold (day 28) higher hepatic gene transfer efficiency. To further corroborate this, we estimated the donor cell Y chromosome copies in the liver of recipient female mice. Our data revealed a 12.7-fold increase in average genome copies of male MSCs in the livers of recipient mice with injury compared to control, 60 days after transplantation. However, in vivo administration of AAV-transduced MSCs in the presence of neutralization antibodies (intravenous immunoglobulin, IVIG) was not beneficial. This is possibly due to the clearance of transplanted MSCs by circulating IVIG and underscores the need to develop suitable in vivo models to study such a mode of gene transfer. Copyright © 2015 John Wiley & Sons, Ltd.

Pancreatic metastasis from renal cell carcinoma 16 years after nephrectomy: a case report and review of the literature.

Two percent of all malignant pancreatic tumors are metastases from other primaries, with small cell lung cancer, colorectal cancer, breast cancer and hematological neoplasms being the commonest. Renal cell carcinoma (RCC) metastasizing to the pancreas is rare and occurs in 2.8% of patients with metastatic RCC. However, RCC is the most common primary leading to solitary pancreatic metastasis. Metastases often present many years after nephrectomy for primary RCC (median time of 8 years) and should therefore be looked for on surveillance or when patients present with upper abdominal symptoms. Complete surgical resection when possible offers the best chance for cure.

Schwannoma of bile duct--a case report.

This is a case report of a 29 year old woman who presented with painless and progressive obstructive jaundice. Imaging investigations of the abdomen revealed a tumour of the common bile duct. She was treated by complete excision of the bile duct and hepaticojejunostomy. The histopathology report of the tumour read as benign schwannoma.

Primary oral tuberculosis: report of two cases.

Oral lesions of tuberculosis though uncommon, are seen in both the primary and secondary stages of the disease. In secondary tuberculosis, the oral manifestations may be accompanied by lesions in the lungs, lymph nodes, or in any other part of the body and can be detected by a systemic examination. Primary oral tuberculosis may present as a diagnostic challenge for the clinician. Here we report two patients with primary tuberculosis in the oral cavity who presented to the dental department, were diagnosed and referred for medical management.

Ichthyosis prematurity syndrome caused by a novel missense mutation in FATP4 gene-a case report from India.

Ichthyosis prematurity syndrome (IPS) is reported mainly from Scandinavia where most of the cases are homozygous or compound heterozygous for the nonsense mutation c.504C>A (p.Cys168*) in exon3 indicating a common ancestor for this mutation. The occurrence of IPS in an Indian patient suggests that it is more widespread than previously reported.

Retroperitoneal leiomyosarcoma infiltrating the inferior vena cava (IVC)--a case report.

Retroperitoneal soft tissue sarcomas are difficult to treat because the retroperitoneal organs and great vessels are often involved by the time the patients come to a surgeon. We present the case of a 48 year old woman with a retroperitoneal leiomyosarcoma that had infiltrated the IVC and the renal veins.

Spontaneous development of neoplasms in severe combined immunodeficient mice.

Severe combined immunodeficient (SCID) mice lack functional T and B cells. This renders them useful for implantation of human cells. The absence of immune cells, however, makes severe combined immunodeficient mice highly susceptible to infections and spontaneous development of malignancies; 2 of 114 CB17/Icr-Prkdc(scid) /IcrIcoCrl severe combined immunodeficient mice aged 9 and 10 months developed spontaneous acute leukaemia and thymic lymphoma. The differential diagnosis of such an atypical lymphoid infiltrate includes 'leaky' severe combined immunodeficient mice, thymic lymphoma and acute leukaemia. Until this time, the link between the development of neoplasms in severe combined immunodeficient mice and the mutation remains unclear.

Vascular diseases await translation of blood vessels engineered from stem cells.

The discovery of human induced pluripotent stem cells (hiPSCs) might pave the way toward a long-sought solution for obtaining sufficient numbers of autologous cells for tissue engineering. Several methods exist for generating endothelial cells or perivascular cells from hiPSCs in vitro for use in the building of vascular tissue. We discuss current developments in the generation of vascular progenitor cells from hiPSCs and the assessment of their functional capacity in vivo, opportunities and challenges for the clinical translation of engineered vascular tissue, and modeling of vascular diseases using hiPSC-derived vascular progenitor cells.

The matrix protein Fibulin-5 is at the interface of tissue stiffness and inflammation in fibrosis.

Fibrosis is a pervasive disease in which the excessive deposition of extracellular matrix (ECM) compromises tissue function. Although the underlying mechanisms are mostly unknown, matrix stiffness is increasingly appreciated as a contributor to fibrosis rather than merely a manifestation of the disease. Here we show that the loss of Fibulin-5, an elastic fibre component, not only decreases tissue stiffness, but also diminishes the inflammatory response and abrogates the fibrotic phenotype in a mouse model of cutaneous fibrosis. Increasing matrix stiffness raises the inflammatory response above a threshold level, independent of TGF-ß, to stimulate further ECM secretion from fibroblasts and advance the progression of fibrosis. These results suggest that Fibulin-5 may be a therapeutic target to short-circuit this profibrotic feedback loop.

An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis.

Subcutaneous xenografts have been used for decades to study hepatocellular carcinoma (HCC). These models do not reproduce the specific pathophysiological features of HCCs, which occur in cirrhotic livers that show pronounced necroinflammation, abnormal angiogenesis and extensive fibrosis. As these features are crucial for studying the role of the pathologic host microenvironment in tumor initiation, progression and treatment response, alternative HCC models are desirable. Here we describe a syngeneic orthotopic HCC model in immunocompetent mice with liver cirrhosis induced by carbon tetrachloride (CCl4) that recapitulates key features of human HCC. Induction of substantial hepatic fibrosis requires 12 weeks of CCl4 administration. Intrahepatic implantation of mouse HCC cell lines requires 30 min per mouse. Tumor growth varies by tumor cell line and mouse strain used. Alternatively, tumors can be induced in a genetically engineered mouse model. In this setting, CCl4 is administered for 12 weeks after tail-vein injection of Cre-expressing adenovirus (adeno-Cre) in Stk4(-/-)Stk3(F/-) (also known as Mst1(-/-)Mst2(F/-); F indicates a floxed allele) mice, and it results in the development of HCC tumors (hepatocarcinogenesis) concomitantly with liver cirrhosis.

Back to the future: examining type 2 diabetic vasculature using the gestational diabetic placenta.

Understanding the association between the intrauterine hyperglycemic milieu and the development of adult diabetic vasculopathy is of particular relevance in India, where diabetes and vascular disease are prevalent. The gestational diabetes mellitus placenta is a valuable tool to examine blood vessels that have been exposed to hyperglycemic cues. We report an interesting observation in a cohort of gestational diabetes mellitus foetal placental vasculature from South India. Transmission electron microscopy demonstrated pericyte detachment and pericyte ghost cells reminiscent of adult type 2 diabetic retinopathy, in gestational diabetes mellitus foetal placental blood vessels that were not observed in non-gestational diabetes mellitus placentas (p ≤0.001). Endothelial cell irregularity was observed in 76% gestational diabetes mellitus foetal blood vessels as compared with 10.4% non-gestational diabetes mellitus placental vasculature (p ≤0.001). Other abnormalities noted in gestational diabetes mellitus placenta included mitochondrial abnormalities, increased micro vessel density and thickening of basement membranes. These results suggest that adult type 2 diabetic vasculopathy has developmental origins in utero.

Improved adeno-associated virus (AAV) serotype 1 and 5 vectors for gene therapy.

Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1-10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia.

Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo.

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Bioengineering of AAV2 capsid at specific serine, threonine, or lysine residues improves its transduction efficiency in vitro and in vivo.

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (~63-90%) than the AAV2-WT vectors (~30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ~8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.