Daniel Martineau

Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo

Bone marrow stromal cells (MSCs) are pluripotent cells capable of differentiation into several tissue types. This present study was performed to determine their functional neoangiogenic potential in vivo. Whole bone marrow was harvested from C57Bl/6 mice, and the adherent cellular fraction was culture expanded for 14 doublings. These MSCs were resuspended in Matrigel and their angiogenic effect assessed in isogenic recipients. At 2 weeks postimplantation, the mean vascular density in Matrigel plugs containing 2 × 106 MSCs/ml was 41±5.0 blood vessels (BVs)/mm2 compared to 0.5±0.7 for empty Matrigel (P<0.001). In comparison, Matrigel plugs containing either recombinant murine VEGF 165 at 50 ng/ml or bovine bFGF at 1000 ng/ml generated 21±5 and 11±2.0 BV/mm2, respectively. Arteriogenesis was observed only in the MSC-containing implants. With the use of LacZ retroviral labeling of ex vivo expanded MSCs, we show that ∼10% of LacZ+MSCs differentiated into CD31+ and VEGF+ endothelial cells. More than 99% of the neoangiogenic phenomena arose from recruitment of host-derived LacZnull vascular structures. Neutralizing anti-VEGF antibodies inhibited the MSC-initiated angiogenic response in vivo by 85% (P<0.001). In conclusion, MSCs have the ability to effectively recruit and participate in angiogenesis and arteriogenesis de novo and VEGF plays a central role in the observed host-derived angiogenic response. We propose that ex vivo expanded autologous MSCs may serve as cell therapy to promote therapeutic angiogenesis.

Levels of organochlorine chemicals in tissues of beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Québec, Canada

High levels of organochlorine chemicals (OC) were found in the blubber of 26 stranded carcasses of beluga whales from an isolated population in the St. Lawrence Estuary (Québec, Canada). These compounds accumulated with age in both sexes, being consistently more concentrated in male tissue; high and variable concentrations were found in four juveniles. Lower levels in females are best explained through massive transfer to the newborn during lactation, resulting in juvenile OC concentrations equal to or higher than in adult males. Concentrations in the liver and kidney expressed on a lipid basis suggest dynamic OC exchange between tissues. The adipose tissue concentrations reported here were higher or equal to those found In some pinnipeds, in laboratory animals, and in domestic animals with severe reproductive failure. These findings suggest that OC contamination is a major factor in the non-recovery of the St. Lawrence beluga population over the last decades.

Pathology of stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Québec, Canada.

From June 1983 to May 1986, thirteen carcasses of stranded beluga whales from a polluted area of the St. Lawrence River, Canada were necropsied. High performance liquid chromatography was performed on the brains of three other animals to determine concentrations of benzo a pyrene (BaP). Two juvenile animals had severe multisystemic lesions one of which, a severe necrotizing dermatitis, was associated with a Herpesvirus-like particle. Four adults had five varieties of tumours. An adult had a systemic nocardiosis and a juvenile was affected ty a non 0:1 Vibrio cholerae septicemia. High concentrations of BaP adducts were found in the brains which were analyzed. Occurrence of BaP adducts in the brain of three whales of this population coincides with the high incidence of tumours. This and the previous finding of high concentrations of organochlorine in the tissues of these animals suggest an important role of industrial contaminants in the recent decrease of this population.

Toxic Compounds and Health and Reproductive Effects in St. Lawrence Beluga Whales

An epidemiologic study was carried out over a period of 9 years on an isolated population of beluga whales (Delphinapterus leucas) residing in the St. Lawrence estuary (Quebec, Canada). More than 100 individual deaths were aged, and/or autopsied and analyzed for toxic compounds, and the population was surveyed for size and structure. Arctic belugas and other species of whales and seals from the St. Lawrence were used for comparison. Population dynamics: Population size appeared to be stable and modeling showed this stable pattern to result from low calf production and/or low survival to adulthood. Toxicology: St. Lawrence belugas had higher or much higher levels of mercury, lead, PCBs, DDT, Mirex, benzo[a]pyrene metabolites, equivalent levels of dioxins, furans, and PAH metabolites, and much lower levels of cadmium than Arctic belugas. In other St. Lawrence cetaceans, levels of PCBs and DDT were inversely related to body size, as resulting from differences in metabolic rate, diet, and trophic position, compounded by length of residence in the St. Lawrence basin. St. Lawrence belugas had much higher levels than predicted from body size alone; levels increased with age in both sexes, although unloading by females through the placenta and/or lactation was evidenced by overall lower levels in females and very high burdens in some calves. No PCDDs and only low levels of some PCDFs were detected in St. Lawrence belugas, while proportions of toxic non-ortho (coplanar) PCBs were low relative to proportions seen in other species. At least ten different PCB methylsulphone metabolites were detected in St. Lawrence belugas. Levels of B[a]P adducts to DNA in St. Lawrence beluga brain and liver approached those associated with carcinogenis in small laboratory animals. Pathology: St. Lawrence belugas were not emaciated, and major findings were: a high prevalence of tumors (40% of animals) including eight malignant neoplasms; a high incidence of lesions to the digestive system (53%), to the mammary glands (45% of adult females), and to other glandular structures (11%); some evidence of immuno-suppression; frequent tooth loss and periodontitis. Two animals had severe ankylosing spondylosis and another was a true bilateral hermaphrodite. No such lesions were observed in 36 necropsies of Arctic belugas and of seals and cetaceans from the St. Lawrence.

Pathology and toxicology of beluga whales from the St. Lawrence Estuary, Quebec, Canada. Past, present and future

Abstract An indigenous population of 450–500 beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence Estuary has been exposed chronically for more than 50 years to a complex mixture of industrial pollutants including organochlorinated compounds (OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals. From 1983 to 1990, we have necropsied 45 well preserved carcasses out of a total of 120 beluga whales reported dead over this period. Of these 45 animals, nine were affected by 10 malignant neoplasms. Fifteen animals (33%) were affected by pneumonia. Milk production was compromised in eight of 17 mature females (41%), by inflammatory changes (seven animals) and cancer (one animal) which affected the mammary glands. Opportunistic bacteria were found in pure culture, and/or in significant amounts in at least two organs in 20 belugas (44%). The concentrations of both total PCBs and highly chlorinated PCB congeners were much higher in St. Lawrence animals than in Arctic beluga whales. OC-induced immunosuppression has been repeatedly demonstrated in a wide variety of animal species. Therefore, it is probable that the immune functions of St. Lawrence beluga whales are impaired. Benzo[α]pyrene adducts were detected in 10 of the 11 St. Lawrence beluga whales of which tissues (six livers, 10/11 brains) were analyzed by a method based on HPLC. No such adducts were found in four Arctic animals. Since benzo[α]pyrene is one of the most potent chemical carcinogens known to man, these compounds might be responsible for some of the cancers observed in that population. Overall, our findings contrast vividly with those of others who found that cancers are exceedingly rare in free-ranging odontocete populations and that the major causes for mortalities in these populations are bacteria, parasites, and trauma.

Concentrations and chromatographic profile of DDT metabolites and polychlorobiphenyl (PCB) residues in stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada

The concentrations and high resolution gas Chromatographic profiles of DDT metabolites and polychlorobiphenyl (PCB) congeners were determined in blubber, liver, kidney and lung tissue and milk samples of stranded beluga whales (Delphinapterus leucas) collected at localities along the coasts of the Saint Lawrence Estuary, Canada from November 1983 through December 1984. The analyses indicate that the major PCB components of the tissues were 2,2′,5,5′-tétra-, 2,2′,4,4′,5-penta-, 2,2′,3,4,4′,5′-hexa-, 2,2′,4,4′,5,5′-hexa-, 2, 2′,3,3′,4,5-hexa-, 2,3,3′,5,5′,6-hexa-, 2,2′,3,4, 5,5′,6-hepta- and 2,2′,3,4,4′,5,5′-heptachlorobiphenyls. Although the highest organochlorine chemical concentrations were found primarily in the blubber, concentrations of 1.72 Μg/g for PCB and 2.04 Μg/g for ⌆DDT were determined in one milk sample. No correlation was established between PCB,p,p-DDE and ⌆DDT concentrations and the fat content of the kidney liver and lung tissues. The Chromatographie patterns of the PCB congeners were similar from one tissue to another with the exception of the kidney; the profile indicates the retention of PCB congeners which are minor components in the other tissues. Relations of residue concentrations between tissue are described and the significance of congener-specific PCB analysis is discussed in terms of the structureactivity effects on PCB persistence and toxicity.

Neo-Organoid of Marrow Mesenchymal Stromal Cells Secreting Interleukin-12 for Breast Cancer Therapy

Bone marrow-derived mesenchymal stromal cells (MSCs), beneficial for regenerative medicine applications due to their wide differentiation capabilities, also hold promise as cellular vehicles for the delivery of therapeutic plasma-soluble gene products due to their ease of handling, expansion, and genetic engineering. We hypothesized that MSCs, gene enhanced to express interleukin-12 (IL-12) and then embedded in a matrix, may act as an anticancer neo-organoid when delivered s.c. in autologous/syngeneic hosts. We performed such experiments in mice and noted that primary murine MSCs retrovirally engineered to secrete murine IL-12 can significantly interfere with growth of 4T1 breast cancer cells in vivo, with a more substantial anticancer action achieved when these cells are embedded in a matrix. Plasma of mice that received the IL-12 MSC-containing neo-organoids showed increased levels of IL-12 and IFN-gamma. Histopathologic analysis revealed less tumor cells in implants of 4T1 cells with IL-12 MSCs, and the presence of necrotic tumor islets and necrotic capillaries, suggesting antiangiogenesis. We also showed that the anticancer effect exerted by the IL-12 MSCs is immune mediated because it is absent in immunodeficient mice, is not due to systemic IL-12 delivery, and also occurs in a B16 melanoma model. This study therefore establishes the feasibility of using gene-enhanced MSCs in a cell-based neo-organoid approach for cancer treatment.

Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse

Wound repair in horse limbs is often complicated by exuberant granulation tissue, a condition characterized by excessive fibroplasia and scarring and that resembles hypertrophic scars and keloids in man. The aim of this study was to compare microvascular occlusion and apoptosis in wounds of the limb with those of the body, which heal normally. Five 6.25 cm2 wounds were created on both forelimbs and on the body of six horses. One limb was bandaged to stimulate excessive fibroplasia. Weekly biopsies were evaluated histologically and immunohistochemically for mutant p53 protein by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling to localize and quantify apoptosis, and by electron microscopy to measure microvessel luminal diameters. Histologic examination revealed protracted inflammation as well as slowed epithelialization and deficient fibroblast orientation in limb wounds, particularly those with excessive fibroplasia. Microvessels were occluded significantly more often in limb wounds, and the balance of apoptotic signals was altered against apoptosis in the former, although this could not be confirmed quantitatively. Data suggest that microvascular occlusion and a dysregulated apoptotic process may be involved in the excessive accumulation of extracellular matrix within limb wounds. This might provide a basis for the development of targeted therapies to prevent and treat excessive fibroplasia and extensive scarring in horses.

Human marrow-derived mesenchymal stromal cells decrease cisplatin renotoxicity in vitro and in vivo and enhance survival of mice post-intraperitoneal injection

Acute kidney injury (AKI) can occur from the toxic side-effects of chemotherapeutic agents such as cisplatin. Bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated wide therapeutic potential often due to beneficial factors they secrete. The goal of this investigation was to evaluate in vitro the effect of human MSCs (hMSCs) secretome on cisplatin-treated human kidney cells, and in vivo the consequence of hMSCs intraperitoneal (ip) implantation in mice with AKI. Our results revealed that hMSCs-conditioned media improved survival of HK-2 human proximal tubular cells exposed to cisplatin in vitro. This enhanced survival was linked to increased expression of phosphorylated Akt (Ser473) and was reduced by a VEGF-neutralizing antibody. In vivo testing of these hMSCs established that ip administration in NOD-SCID mice decreased cisplatin-induced kidney function impairment, as demonstrated by lower blood urea nitrogen levels and higher survival. In addition, blood phosphorous and amylase levels were also significantly decreased. Moreover, hMSCs reduced the plasma levels of several inflammatory cytokines/chemokines. Immunohistochemical examination of kidneys showed less apoptotic and more proliferating cells. Furthermore, PCR indicated the presence of hMSCs in mouse kidneys, which also showed enhanced expression of phosphorylated Akt. In conclusion, our study reveals that hMSCs can exert prosurvival effects on renal cells in vitro and in vivo, suggests a paracrine contribution for kidney protective abilities of hMSCs delivered ip, and supports their clinical potential in AKI.

Experimental Transmission of a Dermal Sarcoma in Fingerling Walleyes (Stizostedion vitreum vitreum)

Dermal sarcoma is a benign skin tumor of adult walleyes (Stizostedion vitreum vitreum) with a suspected viral etiology. A laboratory study was initiated to determine if the tumor could be experimentally transmitted by inoculating young walleyes with materials prepared from tumors from adult fish. Eighty walleye fingerlings were divided into four groups of 20 fish each. Two groups were inoculated intramuscularly at 4 months of age either with live tumor cells or with cell-free filtrates of sonicated tumor cells. The two other groups were used as controls and were inoculated either with cultured cells from normal walleye fry or with tissue culture media. Neoplasms, similar to the dermal sarcoma affecting adult walleyes, were observed after 4 months only in fingerlings inoculated with cell-free filtrates of sonicated tumor cells. Like the tumor affecting wild adult walleyes, the transmitted tumors were restricted to the dermis and originated from the superficial surface of scales. They never invaded locally and never metastasized. The transmitted tumors differed from tumors of adult walleyes in their severity and the absence of osteoid. The multicentric origin of transmitted walleye dermal sarcoma suggests that the virus spreads systemically and that tumor cells are polyclonal. This successful transmission of the lesion, along with the presence of C-type virus particles budding from tumor cells in two of seven tumor-bearing fingerlings, supports a retroviral etiology.